Immunomodulatory potential of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages.

Triple-negative breast cancer is the main type of breast carcinoma that causes mortality among women because of the limited treatment options and high recurrence. Chronic inflammation has been linked with the tumor microenvironment (TME) in breast cancer progression. Clinacanthus nutans (CN) has gained much attention because of its anticancer properties, but its mechanism remains unclear. We aimed to study the qualitative phytochemical content and elucidate the cytotoxicity effects of CN on human triple-negative breast cancer (TNBC), MDA-MB-231 and human macrophage-like cells such as THP-1 by using sulforhodamine B (SRB) assay. As highly metastatic cells, MDA-MB-231 cells can migrate to the distal position, the effect of CN on migration were also elucidated using the scratch assay. The CN effects on ameliorating chronic inflammation in TME were studied following the co-culture of MDA-MB-231/THP-1 macrophages. The cytokine expression levels of IL-6, IL-1β and tumor necrosis factor-alpha (TNF-α) were determined using ELISA assays. The results showed that both ethanolic and aqueous CN extracts contained alkaloid, phenol and tannin, flavonoid, terpenoid, glycoside and steroid. However, saponin was only found in the aqueous extract of CN. CN was not cytotoxic to both MDA-MB-231 and THP-1 cells. The ability of MDA-MB-231 to migrate was also not halted by CN treatment. However, CN ethanol extract decreased IL-6 at 25 μg/mL (p = 0.02) and 100 μg/mL (p = 0.03) but CN aqueous extract increased IL-6 expression at 50 μg/mL (p = 0.08) and 100 μg/mL (p = 0.02). IL-1β showed decreased expression after treated with CN ethanol and CN aqueous both at 25 μg/mL (p = 0.03). TNF-α were significantly decreased after CN ethanol treatment at concentration 25- (p = 0.001), 50- (p = 0.000) and 100 μg/mL (p = 0.000). CN aqueous extract slightly inhibited TNF-α at all 25-50- and 100 μg/mL (p = 0.001, p = 0.000, p = 0.000, respectively). Overall, CN acts by ameliorating the pro-inflammatory condition in the TME and may be a potential strategy for its anticancer mechanism on highly metastatic breast cancer condition. The major pathways that link both cancer and inflammation were NF-κB and STATs thus further study on the upstream and downstream pathways is needed to fully understand the mechanism of CN extracts in cooling the inflamed TME in breast cancer.

Introduction

Breast cancer is the leading cause of death among women in the world, in which triple-negative breast cancer (TNBC) accounts for approximately 15%–20% of all breast carcinoma [1]. TNBC is characterized by the negative expression of the three main breast cancer biomarkers, namely, estrogen (ER), progesterone (PR) and epidermal-growth-factor-2 (HER2) receptors. This condition is associated with rapid growth, distant metastasis and poor prognosis compared with other breast cancer subtypes [2]. The lack of treatment options for TNBC limits the management of this disease [1].

The tumor microenvironment (TME) is composed of the extracellular matrix (ECM) and numerous types of stromal cells, such as endothelial and immune cells, fibroblasts and adipocytes, which play important roles in tumor progression and the response to treatment [3]. The interaction between cancer and heterogenous cells within the TME is critical for carcinogenesis, because TME is an important facilitator of immune escape and cancer progression [4]. During the transition of in situ to invasive carcinoma, tumor and stromal cells secrete ECM-degrading proteases such as matrix metalloproteinases (MMPs), thus destructing the ECM. ECM degradation enables the tumor cells to invade locally and produce aberrantly secreted proteolytic enzymes, chemokines and cytokines to attract leukocytes, modulate tumor remodeling and increase tumor cell invasion to distant organs, leading to metastasis [3, 5]. Targeting the TME, particularly the immune cells, may revert the immune system into a more anti-tumor state [6] that could be beneficial to patients with TNBC setting.

Clinacanthus nutans (CN) (Acanthaceae), which is locally known as “Belalai Gajah” or Sabah snake grass, has gained much attention because of its anticancer properties [7, 8]. As anticancer remedies, the leaves of this plant are commonly used as water decoction for oral ingestion [9]. CN has been studied for its cytotoxic effect against various cancer cell lines. CN induces significant cell death in some types of cancer cells [7] but not in others [10, 11]. The immunomodulatory effects of CN are related to toll-like receptor-4 (TLR-4) and the reduced cytokine secretion in murine macrophage cells [10]. However, this receptor is not involved in the mechanism of apoptotic cell death induced by the combination of CN with gemcitabine in pancreatic cancer cells [11]. This study aimed to investigate the effect of CN on human metastatic breast cancer cells such as MDA-MB-231 alone and in co-culture with human macrophage-like cells such as THP-1. We hypothesized that CN could inhibit breast cancer progression by ameliorating the TME, especially by reducing the pro-inflammatory cytokine expression between cancer and immune cells.

Materials and methods

Chemicals and reagents

Undenatured ethanol, chloroform and glacial acetic acid were purchased from HmbG chemicals, Hamburg, Germany. Iodine potassium iodide, ferric chloride, sodium hydroxide, sulphuric acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), β-mercaptoethanol in PBS, sullforhodamine B (SRB), phorbol 12-myristate 13-acetate (PMA), trichloroacetic acid (TCA), Trizma base, bacterial lipopolysaccharides (LPS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA. RPMI 1640 (Gibco), 1X antibiotic (Gibco), and foetal bovine serum (FBS) were purchased from Thermo Fischer Scientific, USA.

Plant extraction

CN plant was collected from the TKC Herbal Nursery in Jelebu, Negeri Sembilan, Malaysia (Coordinates: 2.770028, 101.995321). The plant sample was sent to Herbarium Unit, Universiti Kebangsaan Malaysia (UKM) for identification (voucher specimen no: ID029/2020). The botanist, Dr. Shamsul Khamis used microscopy technique for plant identification.

CN leaves extracts were obtained as previously described [8, 12]. Briefly, the plant was harvested and cleaned from dirt and soil by rinsing with tap water. The leaves were separated from the stem and air-dried at room temperature until fully dried. The dried leaves were milled into powder by using a grinder. Uniform particle size of the leaves powder was obtained using 315 mm test sieve (Retsch, Germany). CN leaves powder were kept in an air-tight container in cool and dry place at room temperature for storage.

Extraction was done using maceration technique by which powdered CN leaves was soaked in 100% undenatured ethanol or distilled water at a ratio of 1:50 for three days with frequent agitation in an amber container kept away from light (Fig 1). Then, the extracts were filtered before repeating the maceration process with fresh solvent until the filtrates turns light green. The filtrates were combined and concentrated under reduced pressure drying using a rotary evaporator (Heidolph, Germany) with temperature set at 40°C. The extracts were collected in glass sample container and further dried in fume hood. When the extracts reach constant weight, the yield percentage was calculated using the formula: weight of dried extract (g)/ weight of leaves powder (g) x 100. The extracts were kept at 4°C until use.

Fig 1

CN extraction flow chart.

Qualitative phytochemistry analysis

Qualitative phytochemistry analysis was carried out according to previous studies [13, 14]. All extracts were prepared by stirring 0.5 g of CN extracts in 5 mL of distilled water and then filtered using Whatman filter paper.

Saponin test

CN extracts were dissolved in distilled water and shaken vigorously. Stable foam formation indicated the presence of saponin.

Alkaloid test

Alkaloid compounds were determined using Wagner’s test. Approximately 2 g of iodine granules and 1.27 g of potassium iodide was mixed with 5 mL of distilled water. After the solution was homogenous, distilled water was added to make 100 mL of the solution. A few drops of the Wagner’s solution were added into the CN extracts. The formation of brown or reddish precipitation indicated the presence of alkaloid compounds.

Phenol and tannin test

Two mL of 2% (v/v) ferric chloride solution was mixed with 10 mL of CN extracts. The presence of phenol and tannin was confirmed when the mixture turned from blue-green to black.

Flavonoid test

Two mL of 2% (v/v) sodium hydroxide was added to 10 mL of CN extracts, followed by a few drops of 10% (v/v) sulphuric acid. The transformation from an intense yellow color to colorless indicated the presence of flavonoid compounds.

Terpenoid test

Two mL of chloroform solution was added into 10 mL of CN extracts. Three mL of concentrated sulphuric acid was added carefully to form a layer. Reddish brown coloration at the interface indicated the presence of terpenoid compounds.

Glycosides test

Keller-Kiliani test were carried out to determine the presence of glycosides in CN extracts. Approximately, 5 mg of CN extract were dissolved in 1 mL of glacial acetic acid. A few drops of ferric chloride solution were added. Then, 2 mL of concentrated sulphuric acid were added slowly into the mixture. Reddish-brown layer was formed in between of extract and the sulphuric acid layers. The upper part of the mixture turns bluish green indicates the presence of glycosides.

Steroid test

Two mL of chloroform and sulphuric acid were added to 5 mL of CN extracts on the sidewise of the test tube. Formation of reddish-brown ring in between of chloroform and suphuric acid layers indicates the presence of steroid in the extracts.

HPLC profiling

CN ethanol and aqueous extracts were sent to Forest Research Institute of Malaysia (FRIM) for HPLC profiling. Briefly, the standard compounds which were schaftoside, isoorientin, orientin, isovitexin and vitexin were diluted in methanol to make 1000 μg/mL of stock solution, respectively. Serial dilution of each standard compounds was prepared at concentration range of 31.25–500 μg/mL. Approximately 100 mg of CN extracts were dissolved in 5 mL of methanol and sonicated for 20 minutes. Then, the extracts were filtered using PTFE 0.45 μm syringe filter. The extracts were analyzed using HPLC (WATERS 600 quarternary gradient pump, WATERS 717plus autosampler and WATERS 2996 PDA) and HPLC Kinetex Biphenyl C18 (5μm, 250 mm x 4.6mm). The solvent system used were depicted in Table 1. The flow rate was 1.0 mL/min and the injection volume was 10 μL. The detection and quantification of schaftoside, isoorientin, orientin, isovitexin and vitexin was carried out at 330 nm. The retention time values obtained from the selected standard compounds were presented in Table 2.

Table 1

Gradient solvent system.

Time (min)	% A	% B
	(0.1% formic acid in distilled water)	(Acetonitrile)
0	95.0	5.0
30	81.0	19.0
33	5.0	95.0
38	5.0	95.0
39	95.0	5.0
42	95.0	5.0

Table 2

The name of standards and their retention time values.

Name of standard compound	Retention time
Schaftoside	29.990
Isoorientin	30.150
Orientin	31.351
Isovitexin	34.682
Vitexin	34.761

Cell culture

MDA-MB-231 (ATCC HTB-26) was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, Carlsbad, CA) supplemented with 1X antibiotic (Gibco, Carlsbad, CA), 25 mM HEPES and 5% foetal bovine serum. THP-1 (ATCC TIB-202) monocyte cells were maintained in the same supplemented medium as MDA-MB-231 but with addition of 0.05 mM β-mercaptoethanol in PBS (Sigma-Aldrich, USA). All cells were maintained in an incubator humidified with 5% CO2 at 37°C.

Sulforhodamine (SRB) cytotoxicity assay

SRB assay was carried out as previously described to determine the 50% inhibition concentration (IC50) values of all the compounds [15, 16]. Initially, the cells were seeded in 96-well plates at a density of 2 x 105 cells/mL (MDA-MB-231), followed by incubation overnight to allow the cells to adhere to the bottom of the plates. The MDA-MB-231 cells were treated with CN 50%, 70%, 100% ethanolic or aqueous extracts at the concentration range of 0–2.0 mg/mL in separate 96-well plate, THP-1 monocyte cells, 2 x 105 cells/mL were seeded and treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) to induce the differentiation of monocytes into macrophage-like cells. After 5 days, THP-1 macrophages were treated with CN 50%, 70%, 100% ethanolic or aqueous extracts at concentration range of 0–2.0 mg/mL. After 48 h of CN treatment, the cells were fixed in the plates by using 50 μL of 50% (w/v) trichloroacetic acid solution (Sigma-Aldrich, USA), followed by incubation at 4°C for 1 h. The plates were then washed for five times with tap water and air-dried prior to staining with 100 μL of 0.4% (w/v) SRB staining solution. Further incubation was done for 10 min at room temperature. Subsequently, the plates were washed for three times with 1% (v/v) acetic acid to remove the unbound stains. After air-drying, the wells were added with 200 μL of 10 mM Trizma base (Sigma-Aldrich, USA) and shaken well for 10 min. Finally, the absorbance was measured using iMark™ microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 490 nm. All experiments were carried out in triplicates.

Migration assay

Migration assay were carried out according to previous study [17]. In this experiment, MDA-MB-231 cells were seeded in six-well plates at 1 x 107 cells/well. Straight lines were created using a sterile 200 μL pipette tip once the cells reached 100% confluency. The plate was rinsed with sterile PBS to remove floating cells. Cells were then exposed to 0–2 mg/mL CN extracts diluted in serum-free RPMI 1640 media. The wound conditions were monitored for 24 h. Images were captured using Olympus CKX41 light microscope at 20X magnification.

Co-culture of MDAMB-231/THP-1 macrophage-like cells

Co-culture of MDA-MB-231/THP-1 macrophage-like cells were carried out according to previous studies (Fig 2) [10, 18]. THP-1 monocytes were seeded in the Falcon™ cell inserts (1 μm pore size) at the concentration of 2 x 105 cells/mL. Approximately 100 ng/mL of PMA solution was added into the media and incubated for 5 days to differentiate the monocytes into macrophages. MDA-MB-231 cells were seeded in the Falcon™ companion plate a day before THP-1 incubation with PMA ended. On the fifth day, the THP-1 macrophages and MDA-MB-231 cells were co-cultured in a plate and then treated with CN ethanol or aqueous extracts in serum-free media for 1 h, followed by exposure to 20 ng/mL LPS. Plates were further incubated for 18 h at 37°C in an incubator humidified with 5% CO2. The media from upper and lower chambers were collected, pooled and concentrated using Amicon Ultra-4 (Millipore) for the IL-6, IL-1β and tumor necrosis factor alpha (TNF-α) cytokine assay.

Fig 2

Schematic diagram of the co-culture experiment.

Cytokine analysis

The secreted IL-6 IL-1β and TNF-α in the culture medium were quantitated using specific cytokine ELISA kits for IL-1β (Cat no. EZHIL1β), IL-6 (Cat no. EZHIL6), and TNF-α (Cat no. EZHTNFA) (Merck, MA, USA) according to the manufacturer’s instructions. Briefly, the plate was washed 4 times with 300 μL/well of 1X Wash Buffer. Residual buffer was blotted firmly by tapping the plate upside down on absorbent paper. Approximately, 50 μL of Assay Buffer A was added to all wells. Then, 50 μL of standard protein or samples were added to the appropriate wells. The plate was sealed and incubated at room temperature for 2 hours. After 2 hours, the plate was washed 4 times with wash buffer. The detection antibody solution was added into the well and further incubate for an hour at room temperature. Then, the plate was washed 4 times using wash buffer. The wells were soaked with wash buffer for 30 second during the final wash to minimize the background. Hundred microliters of Substrate Solution F provided in the kit were added into each well and incubated for 15 minutes in the dark. The solution color changed from blue to yellow. Optical density was measured using a microplate reader at 450 and 570 nm.

Statistical analysis

All data were analyzed using SPSS version 20 and were presented as the mean ± SD. All data were checked for normality using Shapiro-Wilks’s test. One-way ANOVA was applied with Tukey’s post hoc test. However, for data that violated the homogeneity of variance assumption, Welch statistical analysis was done with Games-Howell post hoc test. The differences between experimental groups were considered statistically significant at *p≤0.05, **p<0.01, ***p<0.001, or ****p<0.0001.

Result

CN qualitative phytochemistry analysis

Qualitative phytochemistry analysis was carried out on both ethanolic and aqueous CN extracts. The result showed that both ethanolic and aqueous CN extracts indicated the presence of alkaloid, phenol and tannin, flavonoid, terpenoid, glycoside, and steroid compounds. However, only aqueous CN extract contained saponin, as shown in Table 3.

Table 3

CN ethanol and aqueous qualitative phytochemistry analysis.

Compound	Clinacanthus nutans extracts
	Ethanol	Aqueous
Saponin	-	+
Alkaloid	+	+
Phenol and Tannin	+	+
Flavonoid	+	+
Terpenoid	+	+
Glycoside	+	+
Steroid	+	+

+Present; -Absent.

CN extraction yields were calculated as below:

Yield CN ethanol (%) = Extract dry weight (g)/Initial extract weight (g) x 100

= 22.5057g/250g X 100

= 9.0%

Yield CN aqueous = Extract dry weight (g)/Initial extract weight (g) x 100

= 63.531g/250g X 100

= 25.4%

Reference standard compounds schaftoside, isoorientin, orientin, isovitexin and vitexin were prepared in methanol. All reference standard compounds were detected in the chromatogram at 330 nm (Fig 3). HPLC profiling was carried out for both CN ethanol and aqueous extract. From the chromatogram, all compounds were presents in CN ethanol such as schaftoside, isoorientin, orientin, isovitexin and vitexin (Fig 4A). However, these compounds were not detected in aqueous extract (Fig 4B). Schaftoside was the most abundant compound in CN ethanol extract but not in CN aqueous extract (Table 4).

Fig 3

HPLC chromatogram of the reference standard compounds.

Fig 4

HPLC chromatogram. a) CN ethanol and b) CN aqueous extracts.

Table 4

Content of schaftoside, isoorientin, orientin, isovitexin and vitexin in CN ethanol and aqueous extracts.

Compound	Average concentration (ppm)		Average percentage of compound in sample±RSD (w/w)
	CN 100% ethanol	CN Aqueous	CN 100% ethanol	CN Aqueous
Schaftoside	3654.05 ±4.23	ND	0.73±4.23	ND
Isoorientin	1945.22 ± 4.87	ND	0.39±4.87	ND
Orientin	1204.07± 0.49	ND	0.24±0.49	ND
Isovitexin	1285.24±4.99	ND	0.26±4.99	ND
Vitexin	653.99±1.38	ND	0.13±1.38	ND

ND: not detected.

Values are means±RSD, n = 3.

Cytotoxic effect of CN on MDA-MB-231 metastatic breast cancer cell lines and THP-1 macrophages

One of the most fundamental steps in anticancer drug discovery is the screening of the antiproliferative and cytotoxicity activity against cancer cells. The present study showed no IC50, as determined using the SRB assay, indicating that CN does not inhibit half of the viability of human metastatic breast cancer cells, MDA-MB-231 and human macrophage-like THP-1 cells. (Fig 5A and 5B). Both MDA-MB-231 cells and THP-1 macrophage-like cells were treated with both ethanol and aqueous extracts for up to 48 h.

Fig 5

Cytotoxicity of CN extracts on cells.

(a). Cell viability of CN extracts on human metastatic breast cancer cells, MDA-MB-231 (b). Cell viability of CN extracts in human macrophage-like cells, THP-1. Exposure to CN extracts showed no remarked difference on the cell viability of both types of cells.

Effect of CN on MDA-MB-231 metastatic breast cancer cell migration

The ability of breast cancer cells to migrate enables them to undergo metastasis to secondary sites, such as bones and lungs. Considering that CN inhibits the metastasis of cancer cells to other organs, we evaluated the effectiveness of CN in inhibiting the migration of highly metastatic breast cancer cells such as MDA-MB-231. These cells successfully migrated into the scratched areas and were not inhibited by CN treatment up to the highest tested concentration of 2 mg/mL following 24 h of exposure (Fig 6).

Fig 6

CN effect on human metastatic breast cancer cells, MDA-MB-231 migration.

Various types of CN extracts cannot inhibit the migration of MDA-MB-231 cells. Cancer cells were able to migrate and closed the scratched wounds in 24 hours.

Effect of CN on IL-6, IL-1β and TNF-α expression levels

Cytokines are small secreted proteins released by cells that have a specific effect on the interactions and communications between cells. CN 100% ethanol and aqueous extracts were used to investigate the cytokine expression in the media of the co-culture of MDA-MB-231 and THP-1 macrophage-like cells. The present study showed that CN extracts may help in ameliorating the inflammation state between human breast cancer cells, MDA-MB-231 and THP-1 macrophage-like cells. The expression of IL-6 cytokine in the media of MDA-MB-231/THP-1 macrophage co-culture was significantly reduced at 25 and 100 μg/mL but not at 50 μg/mL compared with LPS-treated control (Fig 7A). IL-1β cytokine expression was significantly reduced at 25 μg/mL for both CN ethanol and aqueous (Fig 7B). Moreover, TNF-α expression was significantly reduced after treatment with CN ethanol and aqueous when compared to LPS-treated group. (Fig 7C).

Fig 7

Effect of CN on IL-6, IL-1β, and TNF-α level of expression in MDA-MB-231/THP-1 co-culture.

a) CN ethanol extract decreased IL-6 expression at 25- and 100 μg/mL but increased its expressions after treatment with CN aqueous extract at 50- and 100 μg/mL. b) CN ethanol extract inhibited the IL-1β expression at 25- and 100 μg/mL but not at 50 μg/mL. CN aqueous extract significantly inhibited IL-1β expression at 25μg/mL c) CN ethanol and aqueous significantly decreased TNF-α expression at all tested concentrations. Data was obtained from three independent experiment replicates and expressed as mean ± SD. *p <0.05, **p<0.01, ***p<0.001 and ****p<0.0001 against LPS treatment only.

Discussion

Triple-negative breast cancer (TNBC) account for 10%–20% of all types of breast cancer. Incidentally, this minority type of breast cancer accounts for many recurrent and metastatic cases and breast cancer deaths [19]. The only therapy option for patients with TNBC is cytotoxic chemotherapy [20]. Recently, IMpassion130 trial has brought atezolizumab as the pioneer immunotherapy agent for TNBC, thus changing the cancer treatment paradigm [21]. However, this USA Food and Drug Association (FDA)-approved agent has only been applied to patients with TNBC whose breast cancer expresses programmed death-ligand 1 (PD-L1) [22].

Natural products remain as a tremendous source of anticancer drug leads or compounds [23]. In 2014, 53% of the new compounds approved for cancer treatment are of natural product origin [24, 25].]. Clinacanthus nutans (CN) (Acanthaceae) or Sabah snake grass is a medicinal plant with anticancer effect. The leaves of this plant are consumed as a vegetable or taken as tea because of its common health benefits [26, 27]. It is used as an alternative medicine for insect bites, skin rashes, inflammation, burns and scalds, dysentery, diabetes, herpes infection and cancer [28, 29]. This plant can be found in several South East Asian countries, including Malaysia, Thailand and Indonesia. The vernacular names of CN are “belalai gajah” (Malay), Sabah snake grass (English), you dun cao (Chinese), slaed pang pon (Thai), “dandang gendis” or “kitajam” (Indonesian) [9].

Traditionally, dried CN leaves were made into tea or blended with green apple in plain water as healthy juice. Water, which is the most polar solvent, can extract the highest total phytochemical content from the leaves [30]. Moreover, the drying temperature plays a critical role in the CN phytochemical constituent extraction, especially total phenolic and flavonoid contents. A study found that the leaf extract from CN has higher saponin, alkaloid, steroid and tannin contents than the stem part. CN plants that are exposed to sunlight during cultivation promote higher phenolic content than plant cultivated under the shade [13].

The central tenet of western medicine is that a drug should be composed of well-known chemical component or a pure single compound that is selective and target-specific [31]. Most of the conventional cytotoxic anticancer drugs were discovered through random high-throughput screening of compounds in cell-based cytotoxicity assays [32]. Thus, the National Cancer Institute under the Developmental Therapeutics Program has developed a panel of 60 human tumor cell lines and adapted as one of the most important steps in evaluating new anticancer agent [33]. On the other hand, herbal medicine was dependent on evidence-based approach which accumulated over centuries in the form of traditional medicine or folklore medicine [31, 34]. Herbal medicine comprised of multi-component phytochemicals which identification of its active constituent were difficult [35]. More efforts were needed especially in bioassay experiments to build strong scientific evidences for integration of herbal medicine into mainstream medicine to treat cancer. Perhaps, the achievement of YIV-906 (PHY906), a four-herb formulation to be incorporated as anticancer adjuvant for cancer patients serves as model for other types of herbal plant [36]. In case of CN, elucidation of the its mechanism is of the utmost importance to enable this herbal medicine to be regulated as a drug and differentiate it with the commercial herbal product, hence satisfying the unmet medical need [37].

In this study, the cytotoxic effect of CN was evaluated on human metastatic breast cancer cell line such as MDA-MB-231 cells. Both ethanolic and aqueous CN extracts were not cytotoxic to the MDA-MB-231 cells. Our study was similar to those of Mai et al. [10] and Vajrabaya [38], in which CN was not toxic to mouse macrophage cells, RAW 264.7, mouse fibroblast, L929 and human breast cancer cells such as MDA-MB-231 and MCF-7 [11, 39]. However, in other studies, CN exerts antiproliferative effects towards breast cancer cells, MDA-MB-231 [40, 41], ovarian cancer cells, Hela [42], pancreatic ductal adenocarcinoma, AsPC1, BxPC3 and SW1990 [11], erythroleukemia cells and K562 [7, 43] with very potent anticancer activity and IC50 less than 30 μg/mL. The difference of antiproliferative activity of CN extract against MDA-MB-231 between this experiment and other studies was due to the type of solvent used to prepare the extracts. Both studies by Mutazah et al. [40] and Quah et al. [41] used methanolic CN extracts but our study used ethanol and distilled water to prepare the CN extracts. In terms of plant part, our study was similar to Quah et al. [41] that used CN leaves, however, Mutazah et al. [40] used bark to prepare CN methanolic extract. Other than that, previous study reported that the harvesting age and harvesting frequencies may influence the phytochemical content in CN plant especially shaftoside, isoorientin and orientin [44]. The highest total phenolic content and flavonoids can be obtained from harvesting at week 16 during first harvest. Other factor that might explained the difference of the antiproliferative activity of CN was that there might be variation in the amount of phytochemical content in various CN extracts. According to a study, phytochemical production in CN plant was high until 6 months of age and decreased until reaching one year of age [45] and increased harvesting frequency results in decreased amount of total phenolic and flavonoids contents in CN plant [44].

Considering that most of the natural products are screened for their antiproliferative activity before being further studied for their anticancer potential, researchers may have missed several potential agents that may not be cytotoxic but can halt carcinogenesis or metastasis through non-cancer cell death-induced mechanism, such as targeting angiogenesis [46], re-educating the immune cells [47] or reducing the smoldering inflammation in the TME [48, 49]. Therefore, the minimum criteria that must be satisfied in the early phase of pre-clinical anticancer drug development process should not be solely based on the cytotoxic parameters, especially for natural product-based agents.

Inflammation is essential to protect the body from virus or bacterial infections. For example, pro-inflammatory endogenous cytokines such as IL-1, TNF-α, and IL-6 are crucial for the resolution of acute inflammation [50]. However, prolonged inflammation or chronic inflammation can positively influence breast cancer growth and progression [5]. High levels of innate cytokines, as evident in chronic inflammation, can promote tumor progression by inducing the sustained activation of NF-κB [51]. The persistent activation of the immune system in chronic inflammation environment enhances genomic lesions and promotes tumor growth [52]. Pleiotropic IL-6 may play a critical role in the communication between cancerous and non-cancerous cells within the TME [53, 54]. IL-6 is a potent cancer cell growth factor that can induce an epithelial–mesenchymal transition phenotype in breast cancer and therapeutic resistance in breast cancer [55, 56]. IL-1β plays a pivotal role in promoting inflammation in TME, angiogenesis and immunosuppression [57]. The pro-inflammatory factors of IL-1 and IL-6 from TAMs facilitate the invasion of cancer cells, and this process might be associated with their receptor up-regulation [58]. These factors are also involved in the Jak-STAT3 signaling pathway in the chronic inflammation-associated tumor growth and immunosuppressive process [58]. The secreted IL-1β into the TME promotes tumorigenesis, tumor invasiveness and immunosuppression [59]. IL-6 is involved in immune evasion process and T-cell-mediated cytotoxicity [60]. Blocking the IL-1β/IL-6 network in TME could halt tumor progression [61].

Our results showed that 25 and 100 μg/mL ethanolic CN extract suppressed the secretion of IL-6 and IL-1β in the co-culture between human TNBC cell line, MDA-MB-231 and human macrophage-like cells such as THP-1 (Fig 7A and 7B). By contrast, CN aqueous extract increased the secretion of IL-6 at 50 and 100 μg/mL but not of IL-1β at 25 and 50 μg/mL. LPS-induced production of pro-inflammatory cytokines were regulated by multiple pathways such as Nrf2/Keap1 and NF-κB pathways thus overlapping modulation of these pathways may have occurred at certain CN concentration as previously shown by other plant phytochemicals [62]. Other than that, the contribution of CN antioxidant effect may have altered the pro-inflammatory cytokine milieu in the co-culture [63, 64]. High IL-6 expression in co-culture experiment may be due to the high basal IL-6 secretion from MDA-MB-231 cells besides production of LPS-induced pro-inflammatory cytokines from THP-1 macrophages [65]. These results suggested that CN extracts were able to ameliorate the inflammation state in the TME via inhibition of IL-6 or IL-1β secretions from the cancer cells and immune cell interactions at certain concentrations.

Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that mainly mediates anti-tumor effects, but this cytokine can also promote tumor progression [66]. The present study showed that the exposure of the CN ethanolic and aqueous extracts suppressed TNF-α cytokine expression. Previous study also showed that various types of CN extracts significantly reduced the LPS-induced TNF-α production of macrophage cells such as RAW 264.7 [10]. The TNF-α involvement in the development of cancer is complex, and the balance between the high and low levels of TNF has an adverse effect on tumor growth [67]. Activated macrophages were the main cells that produced TNF-α in the TME [68]. Studies also showed that this cytokine was the essential effector cytokines that initiate and maintain chronic inflammation in mouse models [69]. Although TNF-α showed pro-apoptotic functions in tumor cells, most animal models and clinical studies revealed the pro-neoplastic functions of this cytokine [70, 71]. On the other hand, high levels of TNF-α are associated with cancer progression and metastasis [72]. The ability of CN ethanol extract to suppress TNF-α may help to ameliorate the inflamed interaction between breast cancer cells, MDA-MB-231 and macrophage, THP-1.

Breast cancer is not a stand-alone proliferative disease but involved the cooperation between various types of immune cells in the tumor microenvironment [71]. The communications between cancer cells and immune cells were mediated by cytokines by which the cancer cells often take control in shaping the conducive microenvironment for its survival [73, 74]. The conducive microenvironment for cancer cells survival and progression were characterized by the phenomenon of chronic inflammation. Pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6, TNF-α were constitutively expressed in tumor microenvironment to facilitate carcinogenesis [5, 75]. In the current study, CN extracts were not affecting the proliferation and migration of cancer cells but suppressed the pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. This showed that amelioration of the pro-inflammatory cytokines in the tumor microenvironment were the key attributes of the anticancer properties of CN extracts.

Conclusions

In conclusion, CN ethanol and aqueous extracts did not affect the viability of both human breast cancer cells such as MDA-MB-231 and THP-1 macrophage-like cells and was not able to inhibit the migration of cancer cells in vitro. However, the ethanolic CN extracts prevented the smoldering inflammation between cancer cells and immune cells by reducing the level of pro-inflammatory cytokines, such as IL-6, IL-1β and TNF-α. By contrast, the effects of aqueous CN extract on the pro-inflammatory cytokines were limited to IL-1β. Hence, the possible anticancer mechanism through which CN may exert its effect is by reducing the inflammation state of TME. Further research is needed especially on the downstream consequences of the pro-inflammatory cytokines inhibition by CN extracts, as well as the upstream pathways that promote cytokine production such as NF-κB and STATs pathways.

11 Dec 2020

PONE-D-20-27749

Immunomodulatory potentials of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages

PLOS ONE

Dear Dr. Rajab,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

A number of significant issues were raised by the reviewers, one of whom suggested rejection.  Nevertheless, if you feel that you can address these issues we would be willing to consider a revised manuscript.

Please submit your revised manuscript by Jan 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional information about each of the cell lines used in this work, including the source, and any quality control testing procedures (authentication, characterisation, and mycoplasma testing).

For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Kindly mentioned important statistical values inside the abstract.

Further study is needed to fully understand the potential of CN for cancer treatment. Kindly mentioned what further study will be required.

Kindly mentioned source of chemicals and list of equipment. Did you dry the plant?

Please write complete methodology including Plant extraction. This is incomplete. Please include a flow diagram of your extraction procedure.

Did you grind the plant? What is the sieve size?

Is this your new method on Cell culture,Sulforhodamine (SRB) cytotoxicity assay, Migration assay, Co-culture of MDAMB-231/THP-1 macrophage-like cells and Cytokine analysis. Please cite proper reference if you have adapted the methodology.

Please mentioned all abbreviation at the end.

Did you calculate Correlation coefficient, linearity, Limit of detection (LOD) and Limit of quantification (LOQ) for HPLC study. I recommend to include this.

According to your discussion "Our study was similar to

those of Mai et al. 2016 and Khoo et al. 2018, in which CN was not toxic to mouse

macrophage cells, RAW 264.7, mouse fibroblast, L929 [31] and human breast cancer cells

such as MDA-MB-231 and MCF-7 [11, 32]. However, in other studies, CN exerts

antiproliferative effects towards breast cancer cells, MDA-MB-231 [33, 34], ovarian cancer

cells, Hela [35], pancreatic ductal adenocarcinoma, AsPC1, BxPC3 and SW1990 [11],

erythroleukemia cells and K562 [7,36] with very potent anticancer activity and IC50 less than

30 µg/mL."

Some studies showed high activity towards breast caner cell. Why your results does not toxic to mouse

macrophage cells, RAW 264.7, mouse fibroblast, L929 and human breast cancer cells

such as MDA-MB-231 and MCF-7. Please add one paragraph and justify. You have cited the reference but it is not sufficient to proceed. Please compare the methodologies of previous work. You have stated the mechanism as

"Hence, the possible anticancer mechanism through which CN may exert its effect is by

reducing the inflammation state of TME"

If you elaborate this statement in the discussion then your paper will be a better to understand and this is what researchers are looking at.

Please improve your discussion in this context.

Please provide future direction inside the conclusion.

Reviewer #2: Triple negative breast cancer is a difficult to treat phenotype that definitely warrants further investigation. Research into natural ‘local’ remedies to underpin the scientific basis for any effects is interesting and this paper aims to link the two fields by investigating the effects of a well known SE Asian medicinal plant C.Nutans on a triple negative breast cancer cell line and a macrophage cell line in vitro.

Crude extracts from plants contain many phytochemicals, as indicated by the qualitative observation of a wide range of constituents (table 1 ie containing Saponin, Alkaloid, Phenol and Tannin, Flavonoid, Terpenoid, Glycoside, Steroid). HPLC demonstrated a number of flavones and one flavonoid (schaftoside) in the ethanol extract, as may be expected. It isn’t possible to evaluate what the active components are, and whether they could be reproduced by a single purified entity (eg after full characterization by spectral techniques (eg MS, NMR, IR, etc). I am slightly unclear whether this manuscript is about drug discovery (potentially novel compounds from the plant) or herbal medicine, since both are mentioned.

The ethanol and aqueous extracts did not impact on cell viability, and the concept of cancer as a proliferative disease is now old fashioned in the era of immunotherapy. However the authors make a valid point that only screening for agents which inhibit the proliferation of tumour cell lines will miss compounds that effect different pathways. The scratch assay indicated that the extracts did not effect migration either.

It was interesting that the authors included a co-culture experiment to evaluate the effects of the extract on cytokine production. THP-1 are a monocytic leukemia cell line and were differentiated to macrophages using PMA. However, this does not mean they have the characteristics of M2 macrophages (as indicated in the discussion). Other authors consider THP-1 as a good model of M0/M1 differentiation and there are publications using IL4 and IL13 to drive M2 differentiation eg Genin, M., Clement, F., Fattaccioli, A. et al. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 15, 577 (2015). https://doi.org/10.1186/s12885-015-1546-9

I think more specific detail on the co-culture model would be valuable, the seeding density of THP-1 on inserts was stated, but the seeding density / confluence of MDA-MB231 triple negative breast cancer cell line was not (just that they were cultured for 6 days). Was the extract (1h in serum free medium) placed in the upper chamber, lower chamber or both? The differentiated THP-1 were then primed with LPS (20ng/ml), to stimulate cytokine production – which is more representative of infection and not cancer. 18h later the culture supernatant was removed for cytokine analyses – was this the upper chamber / lower chamber and do the authors think the cytokine was from the macrophages only or there could be any contribution by the epithelial cell line? What is the rationale for including the epithelial cells in the co-culture experiments – what contribution do the authors think they are making?

The cytokine data was presented as % of activation rather than the absolute values. I think it would be interesting to see the amount of cytokine produced by LPS stimulated THP-1 +/- extracts. It isn’t clear in the methods how many replicates and whether the data produced is normally distributed. If it is, then ANOVA is more appropriate than a simple t test.

Ultimately, the authors state that their extracts are anti-inflammatory on the basis of the changes in production of IL6, IL1β and TNFalpha, although the aqueous extract seems to show a slight increase in IL6 and TNFalpha (above 100%). If there was a meaningful effect, then a dose response should be seen (not evident for IL6 and IL-1β with the ethanol extract).

In terms of the questions asked:

I think there is some key information missing from the methods section (particularly on the co-culture model) which is important to include since this is the basis for the key findings of the paper.

In addition, t-test may not be the most suitable statistical assay since it only compares two means. ANOVA would be more appropriate given the number of variables (concentration, extract).

The cytokine data is oddly presented (% activation) rather than concentration values - which is what I would expect to see.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Muhammad Shahzad Aslam

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Review.docx

Click here for additional data file.

4 Apr 2021

Reviewer #1:

1. Kindly mentioned important statistical values inside the abstract.

• The statistical values have been added in the abstract.

2. Further study is needed to fully understand the potential of CN for cancer treatment. Kindly mentioned what further study will be required

• The major pathways that link both cancer and inflammation were NF-κB and STATs thus further study on the upstream and downstream pathways is needed to fully understand the mechanism of CN extracts in cooling the inflamed TME in breast cancer. This has been added in the revised manuscript.

3. Kindly mentioned source of chemicals and list of equipment. Did you dry the plant?

• The source of chemicals and equipment have been included in the revised manuscript. Yes, we dried the leaves at room temperature until the leaves is fully dried.

4. Please write complete methodology including Plant extraction. This is incomplete. Please include a flow diagram of your extraction procedure.

• The methodology section has been added and the flowchart is included in the revised manuscript.

5. Did you grind the plant? What is the sieve size?

• Yes, we grind the leaves into powder and sieve using 315 mm test sieve (Retsch, Germany).

6. Is this your new method on Cell culture,Sulforhodamine (SRB) cytotoxicity assay, Migration assay, Co-culture of MDAMB-231/THP-1 macrophage-like cells and Cytokine analysis. Please cite proper reference if you have adapted the methodology.

The appropriate references have been added in the revised manuscript.

• Sulforhodamine cytotoxicity assay Holbeck et al. 2010, Orellana et al. 2016. https://doi.org/10.21769/bioprotoc.1984

• Migration assay were carried out according to Wang et al. 2019 https://dx.doi.org/10.2147%2FOTT.S199605

• Co-culture of MDAMB231/THP-1 macrophage-like cells experiment was carried out according to previous study by Smith 2015 https://doi.org/10.21769/bioprotoc.1638 and Mai 2016 https://doi.org/10.3389/fphar.2016.00007

• Schematic diagram of the method has been added in the revised manuscript.

• Cytokine analysis were carried out as per manufacturer protocols.

7. Please mentioned all abbreviation at the end.

• The abbreviation has been added in the revised manuscript.

8. Did you calculate Correlation coefficient, linearity, Limit of detection (LOD) and Limit of quantification (LOQ) for HPLC study. I recommend to include this.

• Our CN extracts were sent for HPLC service to Forest Research Institute of Malaysia (FRIM). The institution used in-house HPLC validation method which they cannot make the HPLC validation methods available to their clients. However, the chromatogram of the standards used in the experiment has been added in the revised manuscript.

9. According to your discussion "Our study was similar to

those of Mai et al. 2016 and Khoo et al. 2018, in which CN was not toxic to mouse

macrophage cells, RAW 264.7, mouse fibroblast, L929 [31] and human breast cancer cells such as MDA-MB-231 and MCF-7 [11, 32]. However, in other studies, CN exerts

antiproliferative effects towards breast cancer cells, MDA-MB-231 [33, 34], ovarian cancer cells, Hela [35], pancreatic ductal adenocarcinoma, AsPC1, BxPC3 and SW1990 [11], erythroleukemia cells and K562 [7,36] with very potent anticancer activity and IC50 less than 30 µg/mL."

Some studies showed high activity towards breast cancer cell. Why your results does not toxic to mouse macrophage cells, RAW 264.7, mouse fibroblast, L929 and human breast cancer cells such as MDA-MB-231 and MCF-7. Please add one paragraph and justify. You have cited the reference but it is not sufficient to proceed. Please compare the methodologies of previous work.

• The results justification and method comparison has been added in the discussion section.

“In this study, the cytotoxic effect of CN was evaluated on human metastatic breast cancer cell line such as MDA-MB-231 cells. Both ethanolic and aqueous CN extracts were not cytotoxic to the MDA-MB-231 cells. Our study was similar to those of Mai et al. [10] and Vajrabaya [39], in which CN was not toxic to mouse macrophage cells, RAW 264.7, mouse fibroblast, L929 and human breast cancer cells such as MDA-MB-231 and MCF-7 [11, 40]. However, in other studies, CN exerts antiproliferative effects towards breast cancer cells, MDA-MB-231 [41, 42], ovarian cancer cells, Hela [43], pancreatic ductal adenocarcinoma, AsPC1, BxPC3 and SW1990 [11], erythroleukemia cells and K562 [7,44] with very potent anticancer activity and IC50 less than 30 µg/mL. The difference of antiproliferative activity of CN extract against MDA-MB-231 between this experiment and other studies was due to the type of solvent used to prepare the extracts. Both studies by Mutazah et al. [41] and Quah et al. [42] used methanolic CN extracts but our study used ethanol and distilled water to prepare the CN extracts. In terms of plant part, our study was similar to Quah et al. [42] that used CN leaves, however, Mutazah et al. [41] used bark to prepare CN methanolic extract. Other than that, previous study reported that the harvesting age and harvesting frequencies may influence the phytochemical content in CN plant especially shaftoside, isoorientin and orientin [45]. The highest total phenolic content and flavonoids can be obtained from harvesting at week 16 during first harvest. Other factor that might explained the difference of the antiproliferative activity of CN was that there might be variation in the amount of phytochemical content in various CN extracts. According to a study, phytochemical production in CN plant was high until 6 months of age and decreased until reaching one year of age [46] and increased harvesting frequency results in decreased amount of total phenolic and flavonoids contents in CN plant [45]”

10. You have stated the mechanism as, "Hence, the possible anticancer mechanism through which CN may exert its effect is by reducing the inflammation state of TME"

If you elaborate this statement in the discussion then your paper will be a better to understand and this is what researchers are looking at.

Please improve your discussion in this context.

• The discussion on CN mechanism has been added in the revised manuscript.

“Breast cancer is not a stand-alone proliferative disease but involved the cooperation between various types of immune cells in the tumor microenvironment [76]. The communications between cancer cells and immune cells were mediated by cytokines by which the cancer cells often take control in shaping the conducive microenvironment for its survival [77, 78]. The conducive microenvironment for cancer cells survival and progression were characterized by the phenomenon of chronic inflammation. Pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6, TNF-α were constitutively expressed in tumor microenvironment to facilitate carcinogenesis [5, 79]. In the current study, CN extracts were not affecting the proliferation and migration of cancer cells but suppressed the pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. This showed that amelioration of the pro-inflammatory cytokines in the tumor microenvironment were the key attributes of the anticancer properties of CN extracts.”

11. Please provide future direction inside the conclusion.

• Future direction of the study has been added in the conclusion section.

“Further research is needed especially on the downstream consequences of the pro-inflammatory cytokines inhibition by CN extracts, as well as the upstream pathways that promote cytokine production such as NF-κB and STATs pathways.”

Reviewer #2:

1. Triple negative breast cancer is a difficult to treat phenotype that definitely warrants further investigation. Research into natural ‘local’ remedies to underpin the scientific basis for any effects is interesting and this paper aims to link the two fields by investigating the effects of a well-known SE Asian medicinal plant C.nutans on a triple negative breast cancer cell line and a macrophage cell line in vitro.

Crude extracts from plants contain many phytochemicals, as indicated by the qualitative observation of a wide range of constituents (table 1 ie containing Saponin, Alkaloid, Phenol and Tannin, Flavonoid, Terpenoid, Glycoside, Steroid). HPLC demonstrated a number of flavones and one flavonoid (schaftoside) in the ethanol extract, as may be expected. It isn’t possible to evaluate what the active components are, and whether they could be reproduced by a single purified entity (e.g., after full characterization by spectral techniques (eg MS, NMR, IR, etc). I am slightly unclear whether this manuscript is about drug discovery (potentially novel compounds from the plant) or herbal medicine, since both are mentioned.

• It is about herbal medicine. This plant is widely used as alternative medicine for cancer especially in Malaysia, we sought to investigate the scientific evidence of CN extracts effect in breast cancer. We have added a few lines in the discussion part to clarify it.

“The central tenet of western medicine is that a drug should be composed of well-known chemical component or a pure single compound that is selective and target-specific [32]. Most of the conventional cytotoxic anticancer drugs were discovered through random high-throughput screening of compounds in cell-based cytotoxicity assays [33]. Thus, the National Cancer Institute under the Developmental Therapeutics Program has developed a panel of 60 human tumor cell lines and adapted as one of the most important steps in evaluating new anticancer agent [34]. On the other hand, herbal medicine was dependent on evidence-based approach which accumulated over centuries in the form of traditional medicine or folklore medicine [32, 35]. Herbal medicine comprised of multi-component phytochemicals which identification of its active constituent were difficult [36]. More efforts were needed especially in bioassay experiments to build strong scientific evidences for integration of herbal medicine into mainstream medicine to treat breast cancer. Perhaps, the achievement of YIV-906 (PHY906), a four-herb formulation to be incorporated as anticancer adjuvant for cancer patients serves as model for other types of herbal plant [37]. In case of CN, elucidation of the its mechanism is of the utmost importance to enable this herbal medicine to be regulated as a drug and differentiate it with the commercial herbal product, hence satisfying the unmet medical need [38]”.

2. The ethanol and aqueous extracts did not impact on cell viability, and the concept of cancer as a proliferative disease is now old fashioned in the era of immunotherapy. However, the authors make a valid point that only screening for agents which inhibit the proliferation of tumour cell lines will miss compounds that effect different pathways. The scratch assay indicated that the extracts did not affect migration either.

It was interesting that the authors included a co-culture experiment to evaluate the effects of the extract on cytokine production. THP-1 are a monocytic leukemia cell line and were differentiated to macrophages using PMA. However, this does not mean they have the characteristics of M2 macrophages (as indicated in the discussion). Other authors consider THP-1 as a good model of M0/M1 differentiation and there are publications using IL4 and IL13 to drive M2 differentiation eg Genin, M., Clement, F., Fattaccioli, A. et al. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 15, 577 (2015). https://doi.org/10.1186/s12885-015-1546-9

I think more specific detail on the co-culture model would be valuable, the seeding density of THP-1 on inserts was stated, but the seeding density / confluence of MDA-MB231 triple negative breast cancer cell line was not (just that they were cultured for 6 days).

• MDA-MB-231 cells were seeded at 2x105 cells/well a day before THP-1 incubation with PMA ended.

3. Was the extract (1h in serum free medium) placed in the upper chamber, lower chamber or both?

• CN extracts were added into both upper and lower chambers.

4. The differentiated THP-1 were then primed with LPS (20ng/ml), to stimulate cytokine production – which is more representative of infection and not cancer.

• Persistent inflammation can contribute to carcinogenesis. Interaction between cancer cells and immune cells can increased the pro-inflammatory cytokines in the tumor microenvironment. In this experiment, LPS were added to activate THP-1 macrophages and stimulate pro-inflammatory cytokines productions thus created inflamed microenvironment in vitro. The inflamed microenvironment mimics the chronic inflammation that occurs in cancer disease.

5. 18h later the culture supernatant was removed for cytokine analyses – was this the upper chamber / lower chamber and do the authors thing the cytokine was from the macrophages only or there could be any contribution by the epithelial cell line?

• After 18 hours, culture supernatant from upper and lower chambers were combined and concentrated using Amicon® Ultra-4 centrifugal filter units (Merck). Yes, the epithelial cell line can contribute to the cytokine production. MDA-MB-231 can produced IL-6.

6. What is the rationale for including the epithelial cells in the co-culture experiments – what contribution do the authors think they are making?

• The rationale for including the epithelial cells in the co-culture experiments was to mimic the tumor microenvironment which consist of cancer cells and various types of immune cells such as macrophages. THP-1 macrophages were used to mimic the macrophages in tumor microenvironment. Interactions between cancer cells and macrophages were mediated by the pro- inflammatory cytokines such as IL-1β, IL-6 and TNF-α. These pro-inflammatory cytokines create inflamed microenvironment which support cancer progression.

7. The cytokine data was presented as % of activation rather than the absolute values. I think it would be interesting to see the amount of cytokine produced by LPS stimulated THP-1 +/- extracts. It isn’t clear in the methods how many replicates and whether the data produced is normally distributed. If it is, then ANOVA is more appropriate than a simple t test.

• All data were normally distributed as analyzed using Shapiro-Wilks test. Three different replicates were done. We have reanalysed using ANOVA for IL-1β cytokine data. However, IL-6 and TNF-α data violated the homogeneity of variance (Levene’s test) thus Welch test with Games-Howell post-hoc were carried out. All changes have been included in the revised manuscript.

8. Ultimately, the authors state that their extracts are anti-inflammatory on the basis of the changes in production of IL6, IL1β and TNF�, although the aqueous extract seems to show a slight increase in IL6 and TNF� (above 100%). If there was a meaningful effect, then a dose response should be seen (not evident for IL6 and IL-1β with the ethanol extract).

• CN ethanol extract suppressed IL-6 and IL-1β at 25- and 100 µg/mL. However, CN aqueous extract does not suppress IL-6 but IL-1β at 25 µg/mL. The biphasic cytokine suppression effects occurred at low and high concentrations may be due to overlapping modulation of antioxidant and cytokine inhibitory pathway as reported by many phytochemical based anticancer agents [Jodynis-Liebert & Kujawska 2020. https://doi.org/10.3390/jcm9030718]. Oxidative stress is regulated by Nrf2/Keap1 and NF-κB that is also a redox-regulated transcription factor which also regulates inflammatory response [Speciale et al. 2011. https://doi.org/10.2174/156652411798062395]. As antioxidant activity of CN extracts has been reported by previous studies thus CN extracts may have affected one or both pathways that results in the biphasic dose-response [Che Sulaiman et al. 2015. https://doi.org/10.5897/AJPP2015.4396].

9. I think there is some key information missing from the methods section (particularly on the co-culture model) which is important to include since this is the basis for the key findings of the paper.

• We have amended the co-culture method section and added a schematic diagram of co-culture experiment for better understanding of the method.

10. In addition, t-test may not be the most suitable statistical assay since it only compares two means. ANOVA would be more appropriate given the number of variables (concentration, extract).

• We have reanalyzed the data using ANOVA and Tukey’s post hoc test for IL-1β cytokine data. Welch F test with Games-Howell post hoc analysis were done on IL-6 and TNF-α. These changes have been added in the revised manuscript.

11. The cytokine data is oddly presented (% activation) rather than concentration values - which is what I would expect to see.

• We have changed the cytokine data to concentration values (pg/mL). This change is included in the revised manuscript. The revised cytokine graphs have been added in the revised manuscript.

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

24 May 2021

PONE-D-20-27749R1

Immunomodulatory potentials of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages

PLOS ONE

Dear Dr. Rajab,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Several significant issues remains with respect to your manuscript. Reviewer 2 is concerned on two points, namely, can a “three” point curve really be called “biphasic”? and second, have the authors over interpreted their data with respect to actual in vivo tumors?

Please submit your revised manuscript by Jul 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please check all reference before final submission. It is also recommended to avoid any literature that is retracted.

Reviewer #2: CN is an interesting plant and it’s ethnobotanic historic uses certainly recommend further investigation of it’s medicinal properties.

The authors isolate phytochemicals from CN leaves using ethanol / aqueous methods and partially characterize their contents using a range of qualitative tests plus HPLC. The extracts are not cytotoxic and do not induce migration in a scratch assay.

It is an interesting observation that 1h pre-treatment with CN extracts can affect LPS-induced pro-inflammatory cytokine production from human cancer cell lines ie THP-1 macrophage / MDA-MB-321 epithelial co cultures in vitro.

The authors demonstrated that ethanol extracts decreased IL6 production at 25µg/ml and 100µg/ml but not 50µg/ml, however IL6 levels were significantly *increased* with 50µg/ml and 100 µg/ml of aqueous extracts. A similar mixed pattern was seen with IL-1β production in response to the ethanol extracts (ie decreased at 25µg/ml and 100µg/ml but not 50µg/ml). Aqueous extracts inhibited IL-1β production at the two lower concentrations. CN extracts (both ethanolic and aqueous) significantly inhibited TNF alpha production at all the concentrations tested. I presume that the assay conditions were optimized, but it would be interesting to see dose responses over time. Both cell lines are known to express TLR4, which I guess justifies the use of LPS in this model. I am not sure 3 data points (low, high, low) can be reliably called ‘biphasic’ without further information.

Whilst it is good to mimic a tumor micro-environment in vitro, care has to be taken when extrapolating findings and this manuscript has gone too far in interpreting these observations as representing an 'anti-cancer' effect. I disagree that cultured THP-1 represent M2 macrophages, and they are considered more like M1 in the literature (as previously stated).

Reviewer #3: The article by Nordin et al evaluates the effects of Clinacanthus nutans on the co-culture of MDA-MB-231 cancer cells and human THP-1 macrophages, by looking at its effects on the viability, migratory and inflammatory milieu of the co-culture.

The manuscript is well-written and conclusions are drawn appropriately given the supporting evidence.

The authors have responded appropriately to the recommended revisions given by the previous two reviewers.

Minor comments:

• Introduction section “the leaves of this plant are commonly used as water decoction for oral ingestion or soaked in alcohol for topical application to the affected area [9]”

‘the affected area’ should have been clarified.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Muhammad Shahzad Aslam

Reviewer #2: No

Reviewer #3: Yes: Liyana Ahmad

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Jul 2021

Notes for reviewer’s comments are as below and included in the uploaded revised manuscript.

Reviewer #1:

1. Please check all reference before final submission. It is also recommended to avoid any literature that is retracted.

• Authors have checked all references and make sure that there is no retracted literature included in the manuscript to date. Five references have been removed and 2 newly added references has been updated in the list.

Reviewer #2:

1. CN is an interesting plant and it’s ethnobotanic historic uses certainly recommend further investigation of it’s medicinal properties.

The authors isolate phytochemicals from CN leaves using ethanol / aqueous methods and partially characterize their contents using a range of qualitative tests plus HPLC. The extracts are not cytotoxic and do not induce migration in a scratch assay.

It is an interesting observation that 1h pre-treatment with CN extracts can affect LPS-induced pro-inflammatory cytokine production from human cancer cell lines ie THP-1 macrophage / MDA-MB-321 epithelial co cultures in vitro.

• Yes. Similar observations can be obtained from the study by Mai et al. 2016 (https://dx.doi.org/10.3389%2Ffphar.2016.00007). They showed that an hour pre-treatment with CN extract can affect the pro-inflammatory cytokines expression in mouse macrophages RAW 264.7 cells.

2. The authors demonstrated that ethanol extracts decreased IL6 production at 25µg/ml and 100µg/ml but not 50µg/ml, however IL6 levels were significantly *increased* with 50µg/ml and 100 µg/ml of aqueous extracts. A similar mixed pattern was seen with IL-1β production in response to the ethanol extracts (ie decreased at 25µg/ml and 100µg/ml but not 50µg/ml). Aqueous extracts inhibited IL-1β production at the two lower concentrations. CN extracts (both ethanolic and aqueous) significantly inhibited TNF alpha production at all the concentrations tested. I presume that the assay conditions were optimized, but it would be interesting to see dose responses over time. Both cell lines are known to express TLR4, which I guess justifies the use of LPS in this model. I am not sure 3 data points (low, high, low) can be reliably called ‘biphasic’ without further information.

• Authors have removed the biphasic effect from the discussion part. The corrections have been included in the revised manuscript.

“Our results showed that 25 and 100 µg/mL ethanolic CN extract suppressed the secretion of IL-6 and IL-1β in the co-culture between human TNBC cell line, MDA-MB-231 and human macrophage-like cells such as THP-1 (Figure 7a and 7b). By contrast, CN aqueous extract increased the secretion of IL-6 at 50 and 100 µg/mL but not of IL-1β at 25 and 50 µg/mL. LPS-induced production of pro-inflammatory cytokines were regulated by multiple pathways such as Nrf2/Keap1 and NF-κB pathways thus overlapping modulation of these pathways may have occurred at certain CN concentration as previously shown by other plant phytochemicals [62]. Other than that, the contribution of CN antioxidant effect may have altered the pro-inflammatory cytokine milieu in the co-culture [63,64]. High IL-6 expression in co-culture experiment may be due to the high basal IL-6 secretion from MDA-MB-231 cells besides production of LPS-induced pro-inflammatory cytokines from THP-1 macrophages [65]. These results suggested that CN extracts were able to ameliorate the inflammation state in the TME via inhibition of IL-6 or IL-1β secretions from the cancer cells and immune cell interactions at certain concentrations.”

3. Whilst it is good to mimic a tumor micro-environment in vitro, care has to be taken when extrapolating findings and this manuscript has gone too far in interpreting these observations as representing an 'anti-cancer' effect. I disagree that cultured THP-1 represent M2 macrophages, and they are considered more like M1 in the literature (as previously stated).

• Authors agreed to amend the interpretation of the results. Instead of emphasizing on the anticancer effect, we rewrite the discussion part to suggest that CN ameliorates the inflammation condition between cancer cells and macrophages. The appropriate corrections have been included in the revised manuscript.

Reviewer #3:

The article by Nordin et al evaluates the effects of Clinacanthus nutans on the co-culture of MDA-MB-231 cancer cells and human THP-1 macrophages, by looking at its effects on the viability, migratory and inflammatory milieu of the co-culture.

The manuscript is well-written and conclusions are drawn appropriately given the supporting evidence.

The authors have responded appropriately to the recommended revisions given by the previous two reviewers.

Minor comments:

Introduction section “the leaves of this plant are commonly used as water decoction for oral ingestion or soaked in alcohol for topical application to the affected area [9]”

‘the affected area’ should have been clarified.

• Authors decided to amend the phrase, “soaked in alcohol for topical application to the affected area [9]” as it is related to herpes related diseases remedy. We rephrased the statement as “As anticancer remedies, the leaves of this plant are commonly used as water decoction for oral ingestion”.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Jul 2021

Immunomodulatory potentials of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages

PONE-D-20-27749R2

Dear Dr. Rajab,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

2 Aug 2021

PONE-D-20-27749R2

Immunomodulatory potential of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages.

Dear Dr. Rajab:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Salvatore V Pizzo

Academic Editor

PLOS ONE