Anticancer Activity of Mukonal Against Human Laryngeal Cancer Cells Involves Apoptosis, Cell Cycle Arrest, and Inhibition of PI3K/AKT and MEK/ERK Signalling Pathways.

BACKGROUND Laryngeal cancer is one of the major malignancies of the neck and head and is responsible for considerable mortality across the globe. The treatments for laryngeal cancer mainly involve surgical interventions followed by chemotherapy. However, due to unsatisfactory results, constant relapses and the adverse effects associated with the currently used drugs, there is pressing need to develop effective drug options for treatment of laryngeal cancer. Therefore, this study was undertaken to investigate the anticancer effects of a plant-derived alkaloid, Mukonal, against human AMC-HN-8 laryngeal cancer cells. MATERIAL AND METHODS The WST-1 and clonogenic assays were employed to determine the cell viability. Apoptosis was detected by Hoechst and AO/EB staining. Cell migration and cell cycle analysis was performed by Transwell assay and flow cytometry, respectively. Protein expression was examined by Western blotting. RESULTS The results revealed that Mukonal reduced the viability of laryngeal cancer cells dose-dependently. The IC50 of Mukonal was found to be 10 µM. However, the effects of Mukonal on the normal HuLa-PC cells was found to be 140 µM. The decrease in the viability of the AMC-HN-8 laryngeal cancer cells was found to be due to the induction of apoptosis and G2/M cell cycle arrest. Mukonal also suppressed the cell migration and of the AMC-HN-8 laryngeal cancer cells. Mukonal could also inhibit the PI3K/AKT and MEK/ERK signalling pathways in a concentration-dependent manner. CONCLUSIONS Taken together, we conclude that Mukonal could prove a beneficial lead molecule for the treatment of laryngeal cancer.

Background

Cancer is still one of the main challenges to overall public health, and it has been reported that 1 in every 4 deaths in the USA are due to cancer. Many treatments have been tried, but with only very limited success [1]. It is believed that natural metabolites, particularly of plant and microbial origin, may prove beneficial in amelioration of cancers [2]. Several anticancer agents such as vinca alkaloids, podophyllotoxins, and campothecins are of plant origin [3,4]. Among these plant metabolites, alkaloids have been shown to exhibit a wide array of pharmacological properties such as anti-inflammatory, anticancer, and antimicrobial effects [5,6]. Mukonal is an important alkaloid that is mainly isolated from the plant Murraya koenigii [7]. Moreover, total synthesis of Mukonal has also been reported previously [8]. Mukonal has been reported to exhibit antimicrobial and antioxidant activities [9]. However, the anticancer activity of Mukonal has not been thoroughly examined and the antiproliferative effects of Mukonal have not been evaluated against AMC-HN-8 laryngeal cancer cells. In this study, we for the first time report the anticancer activity of Mukonal against AMC-HN-8 laryngeal cancer cells and explore the underlying mechanisms. Laryngeal squamous cell carcinoma (LSCC) is one of the most prevalent malignancies of the head and neck [10]. The currently available treatments strategies include laryngectomy, chemotherapy, and radiotherapy. However, despite these advancements in the treatment of laryngeal cancer, the overall survival rate remains unsatisfactory [11]. In this study we observed that Mukonal decreases the viability of the AMC-HN-8 human laryngeal cancer cells in a dose-dependent pattern. Further, investigation of the underlying mechanism revealed that Mukonal induces apoptosis and G2/M cell cycle arrest in AMC-HN-8 laryngeal cancer cells. The effect of Mukonal was also investigated on cell migration and invasion and it was observed that Mukonal could inhibit both migration and invasion of the laryngeal cancer cells. The MEK/ERK and PI3K/AKT signalling pathways are important pathways that have been reported to be activated in several types of cancers [12]. In the present study we found that Mukonal inhibited both of these pathways in human laryngeal cancer cells. We propose that Mukonal may serve as a lead molecule for the treatment of laryngeal cancer, and synthesis of new and more effective derivatives by synthetic chemistry approaches may prove very beneficial.

Material and Methods

Cell lines and culture conditions

The laryngeal cancer cell line AMC-HN-8 and normal HuLa-PC laryngeal cells were procured from the Shanghai Institutes for Biological Sciences, Chinese Academy of Science. These cell lines were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, antibodies (100 units/mL penicillin and 100 μg/mL streptomycin), and 2 mM glutamine. The cells were cultured in a CO2 incubator (Thermo Scientific) at 37°C with 98% humidity and 5% CO2.

Cell viability and colony formation assays

The cell viability of the laryngeal cancer cells was assessed by WST-1 colorimetric assay. Briefly, the AMC-HN-8 laryngeal cancer cells were seeded in 96-well plates at the density of 2×105 cells/well. The cells were then incubated with WST-1 at 37°C for 4 h. The absorbance at 450 nm was then assessed by a microplate reader to determine the viability of laryngeal cancer cells. To assess the impact of Mukonal on the colony-formation potential of Mukonal, the AMC-HN-8 cells were collected at exponential phase of growth and counted using a hemocytometer. The plating of the cells was carried out at 200 cells/well. The plates were then kept at 37°C for 48 h to permit the cells to adhere. This was followed by the addition of various concentrations (0, 10, 20, and 40 μM) of Mukonal. Following treatment with Mukonal, the cells plates were again incubated for 6 days. After incubating the cells for about 6 days, they were subjected to washing with PBS and fixation with methanol. The AMC-HN-8 cells were then stained with crystal violet followed by microscopy.

Detection of apoptosis

The nuclear morphology of the AMC-HN-8 laryngeal cancer cells was assessed by fluorescence microscopy after subjecting the cells to cell-permeable Hoechst 33342 dye. Ten fields with 100 cells/field were randomly selected for estimation of the cells with condensed nuclei. AO/EB double-staining was used for the determination of apoptotic laryngeal cancer cells as described previously [13].

Cell cycle analysis

To investigate the distribution of the AMC-HN-8 cells in different phases of the cell cycle, approximately 1×105 cells in each well in 6-well plates were kept at 37°C overnight to allow the cells to adhere. This was followed by treatment with various doses of Mukonal (0, 10, 20, and 40 μM) followed by incubation at 37°C. Finally, the distribution of the AMC-HN-8 cells in various cell cycle phases was determined by flow cytometry.

Cell migration assay

The cell migration ability of the laryngeal cancer cells was evaluated by Transwell assay. Briefly, the cells were seeded at 2×105 cells/mL density after 48-h transfection. Thereafter, 200-ml cell suspensions were added into the upper chamber and complete medium was added into the bottom wells. After 24-h culturing, the cells in the upper chambers were removed and cells migrated through the chambers were subjected to fixation with methyl alcohol followed by staining with crystal violet. Finally, the number of cells that migrated was determined by counting the cells under an inverted microscope (magnification 200×, 10 different fields).

Western blot analysis

The laryngeal cancer AMC-HN-8 were lysed with ice-cold hypotonic buffer. After estimating the protein concentrations in each of the cell extracts, the samples containing the proteins were loaded and separated on SDS–PAGE. This was followed by transfer to a nitrocellulose membrane and incubation with the primary antibody (p-PI3K, p-AKT, PI3K, AKT, p-MEK, p-ERK, MEK, and ERK) (1: 1000) for 24 h at 4°C. Then, the membrane was incubated with HRP-conjugated secondary antibody (1: 1000) for at 24°C for about 1 h. The visualization of the proteins was carried out by enhanced chemiluminescence reagent.

Results

Mukonal decreases the viability AMC-HN-8 cell line

To examine the effects of Mukonal (Figure 1A) on the viability AMC-HN-8 laryngeal cancer cell line, the AMC-HN-8 cells were subjected to treatment with Mukonal at varied doses. It was observed that Mukonal displayed significant anticancer effects against AMC-HN-8 with an observed IC50 of 20 μM. The effect of Mukonal on the growth of AMC-HN-8 cells exhibited a concentration-dependent pattern (Figure 1B). However, the effects of Mukonal on the normal HuLa-PC laryngeal cells were less pronounced and Mukonal exhibited an IC50 of 140 μM against them. In addition, we observed that Mukonal treatment caused a considerable reduction in the number of AMC-HN-8 colonies in a concentration-dependent pattern (Figure 2).

Figure 1

A) Chemical structure of Mukonal. (B) Effect of Mukonal on the proliferation of laryngeal cancer AMC-HN-8 and normal HuLa-PC cells. The experiments were repeated 3 times and are presented as mean ±SD (p<0.05).

Figure 2

Effect of Mukonal on the colony formation potential of AMC-HN-8 cells at indicated concentrations. The experiments were repeated 3 times and are presented as mean ±SD (p<0.05).

Mukonal triggers apoptosis in AMC-HN-8 cells

To determine the cause of the anticancer effects, Mukonal-treated AMC-HN-8 cells were subjected to DAPI staining. We observed that Mukonal caused apoptosis in AMC-HN-8 cells in a dose-dependent manner (Figure 3). The analysis of the apoptotic cell population was further confirmed by the AO/EB staining. The apoptotic AMC-HN-8 cells increased with increase in the doses of Mukonal, as obviously indicated by the orange-colored nuclei (Figure 4).

Figure 3

Hoechst staining showing induction of apoptosis in AMC-HN-8 cells upon treatment with Mukonal. The experiments were repeated 3 times and are presented as mean ±SD (p<0.05).

Figure 4

AO/EB staining showing induction of apoptosis in AMC-HN-8 cells upon treatment with Mukonal. The experiments were repeated 3 times.

Mukonal induces G2/M cell cycle arrest in AMC-HN-8 cells

To assess the effect of Mukonal on the distribution of the AMC-HN-8 in different phases of the cell cycle, cells were treated with 0, 10, 20, and 40 μM of Mukonal for 24 h. We found that the percentage of cells at G2 phase increased in a dose-dependent manner, causing cell cycle arrest and leading to G2/M cell cycle arrest (Figure 5).

Figure 5

Mukonal at varied concentrations triggers G2/M cell cycle arrest of AMC-HN-8 cells as indicated by flow cytometry. The experiments were repeated 3 times.

Mukonal inhibits cell migration of AMC-HN-8 cells

The effects of Mukonal on the migration of the laryngeal AMC-HN-8 cancer cells was assessed by Transwell assays. The results showed that Mukonal suppressed the capacity of the AMC-HN6 cells to migrate at IC50 concentration (Figure 6).

Figure 6

Effect of Mukonal on the migration of the AMC-HN-8 cells as revealed by the Transwell assay. The experiments were repeated 3 times and are presented as mean ±SD (p<0.05).

Mukonal inhibits the MEK/ERK and PI3K/AKT signalling pathway

The effect of Mukonal was also examined on the m-TOR/PI3K/AKT signalling pathway. The results revealed that Mukonal inhibited the expression of some of the important proteins of this pathway. We observed that the expression of p-PI3K and p-AKT was significantly downregulated. However, the expression of PI3K and AKT remained almost unaltered (Figure 7). In the MEK/ERK signalling pathway, Mukonal inhibited the expression of p-MEK, p-ERK, MEK, and ERK (Figure 8).

Figure 7

Effect of Mukonal on protein expression of the PI3K/AKT pathway as indicated by Western blotting. The experiments were repeated 3 times.

Figure 8

Effect of Mukonal on the protein expression of MEK/ERK pathway as indicated by Western blotting. The experiments were repeated 3 times.

Discussion

Laryngeal cancer causes significant mortality worldwide and its incidence is growing [14]. The treatment for laryngeal cancer mainly involves laryngectomy followed by chemotherapy or radiotherapy [15]. However, the clinical results for laryngeal cancer are still poor. Chemotherapy is associated with adverse effects and frequent relapses have been observed in laryngeal cancer patients [16]. Moreover, the development chemo-resistance is another obstacle in the treatment of laryngeal cancer. Natural products with less serious adverse effects may prove beneficial in the treatment of cancer [17]. Because many anticancer drugs are natural plant products, plant extracts and their isolated compounds are constantly being screened for their anticancer effects [18]. In this study, we evaluated the anticancer activity of Mukonal against the laryngeal cancer AMC-HN-8 cells. The results revealed that Mukonal dose-dependently reduced the viability of the AMC-HN-8 cells. These antiproliferative effects were further supported by the results of colony formation assay, wherein Mukonal was found to dose-dependently inhibit the viability of AMC-HN-8 cells. Several plant-derived alkaloids have been reported to inhibit the viability of cancer cells, as indicated by previous studies [19]. For instance, the vinca alkaloids have been found to inhibit the viability of a number of cancer cell types. In yet another study, matrine has been reported to inhibit the proliferation of the cancer cells. Previous studies have reported that many of the plant-derived alkaloids exert their anticancer effects by triggering apoptosis [20]. Apoptosis is considered as one of the imperative processes which allows the body to eliminate harmful cells from the body [21]. In the present study, we observed that Mukonal could trigger apoptosis in the AMC-HN-8 cancer cells, as evident from the AO/EB and Hoechst staining results. Another very important mechanism used by anticancer agents is cell cycle arrest. If the cancer cells are arrested in any phase of the cell cycle, they are unable to complete cell division and tumor growth is halted [22]. We observed that Mukonal induced G2/M cell cycle arrest in AMC-HN-8 cells. The effect of Mukonal was also assessed on the migration of the cancer cells and we observed that Mukonal inhibited the migration and invasion of cancer cells. This indicates that Mukonal may prove to be an anti-metastatic agent and may prevent the metastasis of cancer cells in vivo. In most cancers, the expression of a number of pathways and genes is dysregulated. These genes and pathways are considered important therapeutic targets for the treatment of cancers [23]. The PI3K/AKT and MEK/ERK signalling pathways have been found to be upregulated in cancer cells and studies have indicated that these pathways can be targeted for the treatment of cancer [12]. In the present study, we observed that Mukonal inhibited both of these pathways, indicating the anti-proliferative effects of Mukonal. Carbazole alkaloids such as Mukonal are an important class of alkaloids that can be utilized to target these signalling pathways [7]. However, evaluation of Mukonal against more cell lines and in vivo experiments will be required to establish Mukonal as an option for cancer treatment.

Conclusions

We showed that Mukonal inhibits the proliferation of laryngeal cancer cells by prompting apoptosis and G2/M cell cycle arrest. Mukonal also inhibits the migration and invasion of laryngeal cancer cells by targeting the PI3K/AKT and MEK/ERK signalling pathways. As such, Mukonal may prove to be beneficial lead molecule for the treatment of laryngeal cancer and warrants further studies.