The New Compound of (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide to Mediate the Expression of Some Apoptosis Genes by the HepG2 Cell Line

Objectives: Hepatocellular carcinoma is one of the most frequent cancers worldwide, for the treatment of which various therapy protocols and drugs have been introduced; however, none of them has suppressed cancer tissues completely. New research programs have been developed on cancer and the accompanied effects of novel synthesized compounds on cancer cell lines. Our latest reports on the molecular basis of cancer revealed a pattern of changes in gene expression triggered in the cancer pathway.
Methods: HepG2 cell lines were cultured under similar conditions in both test and control groups. The IC50 concentration of the (2R, 4S)-N-(2, 5-difluorophenyl)-4-hydroxy-1-(2, 2, 2-trifluoroacetyl) pyrrolidine-2-carboxamide compound was used in the treatment group. After 48 hours from the culture, the expressional profiles of apoptosis pathway genes (84 genes) were studied using the PCR array method.
Results: The findings demonstrated that the expression of some apoptosis-related genes pertaining to TNF, BCL2, IAP, and caspase families was regulated by (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide. In the same vein, an alteration was observed in the expression of both pro-apoptotic and anti-apoptotic genes associated with the extrinsic and intrinsic apoptosis signaling pathways. Conclusions: According to the data obtained, the pyrrolidine-2-carboxamide compound was demonstrated to be able to regulate the apoptotic activities of HepG2 cells by affecting both pro-apoptotic and anti-apoptotic relevant genes. Creative Commons Attribution License

Introduction

Cells are considered as the basic structural and functional units of human life. Normally, older cells undergo division and proliferate in order to generate newer ones; in addition, they are destroyed as a result of apoptosis in a complicated process, so equilibrium exists between live and dead cells, under normal circumstances (Hanahan and Weinberg, 2000). The malignancy of liver cancer is among the six most frequent cancer types, being considered the third cause of cancer deaths worldwide (Moy et al., 2013). Unfortunately, the incidence rate of the various types of liver cancer has increased in developing countries in recent years. There exists no efficient treatment for liver cancer to date, so the development of advanced treatments for the disease will be highly valued. Accordingly, a wide variety of synthetic pro-apoptotic and anti-apoptotic compounds has been developed for the treatment of various tumors, including liver cancer (Hersey and Zhang, 2001).

N-heterocyclic structures are the major classes of such compounds that play a major role in medicinal chemistry and in particular in drug synthesis (Ten Holte et al., 2001). Organofluorine compounds (organoFs) have been utilized by a large number of pharmaceutical, agrochemical, and drug industries in the past few decades (Hudlicky, 1979). From among them, trifluoroacetimidoyl chlorides substituted molecules have been investigated extensively, with the indication that these fluorinated structures demonstrate interesting biological activities (Polshettiwar and Varma, 2010; Romero et al., 2015).

Based on the introduction above, the authors of this article synthesized the new derivative of (2R, 4S)-N-aryl-4-hydroxy-1-(2, 2, 2-trifluoroacetyl) pyrrolidine-2-carboxamides (Darehkordi and Ramezani, 2017) (Figure 1). Apoptosis is defined as a homeostatic cellular event by which cell populations are preserved in tissues. The apoptosis phenomenon occurs normally as cells proliferate or become aged. Apoptosis is also involved actively in defensive processes, principally in the pathogenesis of a variety of human diseases including, viral infections, autoimmune diseases, and cancer (Norbury and Hickson, 2001). It is well established that some anticancer drugs act by inducing apoptosis in order to exert their cytotoxic effects (Ferreira et al., 2002).

Figure 1

The Chemical Composition Image of (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide (C13H11F5N2O3) (Darehkordi and Ramezani, 2017).

Synthetic compounds which possess cytotoxic properties have apoptosis-inducing potentials in numerous types of human cancer cells. The study of apoptosis-inducing effects under both normal and modified conditions is of high significance (Hersey and Zhang, 2001). Thus, the present study was conducted aimed at investigating the effects of the (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide derivative on cellular apoptosis in human hepatocellular carcinoma cells (HepG2 cells). For this purpose, the authors of this article used RT2 Profiler PCR Arrays to detect the profile of gene expression changes in response to the new derivative of pyrrolidine-2-carboxamide in HepG2 cells.

Materials and Methods

Chemical compound preparation

The powdered sample was provided by dissolving 5mg of the chemical compound in 1mL of DMSO (dimethyl-sulphoxide) to achieve an appropriate concentration of 5mg/mL. Next, the produced stock solution was filtered through a 0.45µM filter, before use in each assay. In the following step, 400µL of the stock solution was mixed up gently and enriched with 600µL of RPMI 1640 at appropriate concentrations (125µg), which was required for examinations by serial dilution (Ramezani et al., 2017).

The cell culture method

A sample of HepG2 cells was provided from the National Cell Bank of Iran (the Pasteur Institute of Iran, Tehran). The cells were cultured in a 25mL culture flask in the RPMI 1640 medium (Gibco, Pasteur Institute, Tehran, Iran). Next, they were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin-streptomycin (100 U/mL) and incubated at 37°C in a humidified atmosphere containing 95% O2 and 5% CO2. As already mentioned in brief, cell growth inhibition (IC50) was calculated to be 50% for pyrrolidine-2-carboxamide after 48 hours (62.5µg/mL) (Ramezani et al., 2017). The nonmalignant cells (L929) were also cultured under similar conditions in the RPMI 1640 medium containing 5% (v/v) of fetal bovine serum (FBS) and 100 units/mL of penicillin-streptomycin. The cultures were left overnight and then incubated at the required concentrations of the pyrrolidine derivative (62.5µg/mL) for 48 hours to be examined by the real-time Profiler PCR Array (The RT2 Profiler PCR Array, SABiosciences, USA).

RT2 Profiler PCR array

The RT2 Profiler PCR Array (SABiosciences, USA) was performed to analyze the expression of a panel of genes involved in the apoptosis pathway (Human Apoptosis PCR Array, RT2 Strand Kit, SABiosciences, USA) together with 5 housekeeping genes. This Real-Time PCR (RT-PCR) kit contained 84 apoptosis-related genes, 5 housekeeping genes, as well as 3 RNA and PCR quality controls. The cells received either a relative concentration of the new therapeutic compound of (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide) or the control medium alone and were kept at 37°C. Following 48 hours from the treatment, the cells were rinsed with PBS and harvested through centrifugation for the purpose of RNA isolation. The total RNA content was extracted using the Qiagen kit (USA) in accordance with the manufacturer’s recommended protocol. The purity and fidelity of the RNA were examined using the spectrophotometric method (by the calculation of the 260/280nm absorbance ratio) and running on the 1.5% agarose gel, with electrophoresis utilized to assess the purity and integrity of RNA. The first strand of cDNA was generated by the RT2 First Strand kit (Qiagen, USA) from 1.0µg of the total RNA for 96-well plates. An ABI, Step 1 plus RT2 PCR Detection System (ABI, Step 1 plus, USA), was applied for the real-time PCR analysis. For each PCR array plate, an experimental cocktail was produced using 998.4µL of the RT2 qPCR master mix, 96µL of the first strand cDNA’s synthesis reaction, and 825.6µL of ddH2 O. The 20µL reaction of the experimental cocktail was aliquoted into each PCR array well. The RT2 qPCR thermal cycling program included one cycle at 95°C for 10min, 40 cycles at 95°C for 15s, 60°C for 40s, and 72°C for 30s (Mirzaei et al., 2014).

Statistical analysis

The authors of this article analyzed the data quantitatively by employing the 2-(∆∆Ct) method, with the threshold cycle (CT) values exported and analyzed using the web-based software of RT2 Profiler PCR Array Data Analysis, version 3.5 (HTTP:// PCR data analysis.sabiosciences.com/pcr/array analysis. PHP). The fold changes of the relevant genes were calculated by the 2-(∆∆Ct) formula. The PCR array was performed in triplicate for each sample, i.e. the extract-treated sample and the control sample.

Results

The effects of pyrrolidine derivatives on apoptosis-related genes

To explore the expression of the critical key genes involved in cellular apoptosis in response to being exposed to the pyrrolidine derivative (0.5 mg/mL) for 24 hours, a quantitative Real-Time qPCR method was utilized by employing the Human Apoptosis RT² Profiler PCR Array. For this purpose, numerous members of gene families, including TNF/TNFR proteins, BCL2 and BAG proteins, BIR proteins, TRAF proteins, and caspases were analyzed. The genes exhibiting fold changes up to two (fold-regulation>2) and less than 0.5 (fold-regulation> -2) were regarded to be expressed differentially (Table 1). Table 3 shows the fold regulation of the examined genes by the PCR array. From among the genes detected, 8 of them were upregulated, yet 14 of them were downregulated (Figure 2). Accordingly, the genes that underwent altered expression included TNF/TNFR family members, such as FASLG, LTA, TNF, TNFRSF10A, and TNFRSF21 (Table 2). The results indicated that the BCL2 and BAGn family members, including BAG4, BAX, BCL2A1, and BCL2L11 also displayed some degrees of changes in their change expression. The BIR family members, such as BIRC3 and XIAP together with TRAF family members were shown to have been altered. However, no expression alteration was observed in the caspase family members, including CASP10, CASP14, CASP3, CASP4, CASP2, and CASP5 (Table 2). The findings in Table 1 demonstrated that in addition to genes, such as BAD (BCL2-associated agonist of cell death) and BAG4 (BCL2-associated athanogene 4), BCL2-like1 and BCL2-like11 (the apoptosis facilitator), Baculoviral IAP repeat containing the 6 (BIRC6), BCL2-interacting killer (apoptosis-inducer), BCL2/adenovirus E1B 19kDa interacting protein 3(BINP3), V-RAF murine sarcoma viral oncogene homolog B1 (BRAF), CASP10, 2, 8, and 14 were downregulated strongly after further treatments. In addition, the tumor necrosis factor-alpha receptor superfamily, member 10a and b (TNFRSF10A, B), CD40 ligand, as well as the Cell death-inducing DFFA-like effector (CIDEA) were shown to have been down-expressed in response to this derivative (Table 1).

Table 1

Gene’s up/down-Regulated after Pyrrolidine-2-Carboxamide Treatment

Gene symbol	Protein/gene name	Activity	Fold changes
AKT1	V-at murine thymoma viral oncogene homolog 1	Anti-apoptosis	42.5
BAG3	BCL2-associated athanogene 3	Anti-apoptosis	6.15
BAX	BCL2-associated X protein	Pro-apoptosis	-7.07
BCL2	B-cell CLL/lymphoma 2	Pro-apoptosis	-9.19
BCL2A1	BCL2-related protein A1	Pro-apoptosis	23.21
BCL2L11	BCL2-like 11 (apoptosis facilitator)	Pro-apoptosis	-17.94
BCLAF1	BCL2-associated transcription factor 1	Pro-apoptosis	5.15
BFAR	Bifunctional apoptosis regulator	Anti-apoptosis	5.56
BID	BH3 interacting domain death agonist	Pro-apoptosis	-4.21
BIK	BCL2-interacting killer (apoptosis-inducing)	Pro-apoptosis	-36.51
BIRC2	Baculoviral IAP repeat containing 2	Anti-apoptosis	30
BIRC3	Baculoviral IAP repeat containing 3	Anti-apoptosis	7.585
BIRC6	Baculoviral IAP repeat containing 6	Anti-apoptosis	-25.17
BNIP1	BCL2/adenovirus E1B 19kDa interacting protein 1	Anti-apoptosis	-8.95
BNIP3	BCL2/adenovirus E1B 19kDa interacting protein 3	Anti-apoptosis	-70.13
BRAF	V-RAF murine sarcoma viral oncogene homolog B1	Anti-apoptosis	-18.48
CASP10	Caspase 10, apoptosis-related cysteine peptidase	Pro-apoptosis	-11.81
Gene symbol	Protein/gene name	Activity	Fold changes
CASP14	Caspase 14, apoptosis-related cysteine peptidase	Pro-apoptosis	-26.86
CASP3	Caspase 3, apoptosis-related cysteine peptidase	Pro-apoptosis	9.57
CASP5	Caspase 5, apoptosis-related cysteine peptidase	Pro-apoptosis	34.33
CASP8	Caspase 8, apoptosis-related cysteine peptidase	Pro-apoptosis	-18.82
CD40LG	CD40 ligand	Anti-apoptosis	-47.94
CIDEB	Cell death-inducing DFFA-like effector b	Pro-apoptosis	-12.94
FASLG	Fas ligand (TNF superfamily, member 6)	Pro-apoptosis	17.42
IGF1R	Insulin-like growth factor 1 receptor	Anti-apoptosis	-9.75
RIPK2	Receptor-interacting serine-threonine kinase 2	Anti-apoptosis	-4.53
TNF	Tumor necrosis factor	Pro-apoptosis	25.99
TNFRSF10A	Tumor necrosis factor receptor superfamily, member 10a	Death domain proteins	-85.66
TNFRSF10B	Tumor necrosis factor receptor superfamily, member 10b	Death domain proteins	-74.28
TNFRSF1A	Tumor necrosis factor receptor superfamily, member 1A	Death domain proteins	-5.2
TNFSF10	Tumor necrosis factor (ligand) superfamily, member 10	Pro-apoptosis	-23.63
TP53	Tumor protein p53	Pro-apoptosis	36.38
TP73	Tumor protein p73	Pro-apoptosis	-4.19

Figure 2

The Scatter Plot Created in the PCR Array Data Analysis Web Portal Compares the Normalized Expression of Each Gene on PCR Array between 2 Groups. The upper left section (red dots) indicates upregulated genes, while the lower right section (green dots) indicates downregulated genes and the black dots indicates unchanged expressions of the genes.

Table 2

A Complete List of Apoptotic Genes’ Expression Alteration in HepG2 Cell Line

No	Regulation intensity	Position in PCR panel	Gene symbol	fold regulation
1	↑↑↑↑↑	A02	AKT1	42.5
2	↑↑	A06	BAG3	6.157
3	↑↑↑↑↑	A12	BCL2A1	23.21
4	↑↑	B02	BCL2L10	2.9737
5	↑↑	B04	BCL2L2	2.5396
6	↑↑	B05	BCLAF1	5.5132
7	↑↑↑↑↑	B10	BIRC2	30.0046
8	↑↑	B11	BIRC3	7.585
9	↑↑	C08	NOD1	2.399
10	↑↑	D03	CASP3	9.577
11	↑↑↑↑↑	D05	CASP5	34.33
12	↑↑	D07	CASP7	3.6785
13	↑↑	D10	CD40	3.9391
14	↑↑↑↑	E08	FASLG	17.423
15	↑↑↑↑↑	F06	TNF	25.9996
16	↑↑↑↑↑	G06	TP53	36.3841
17	↑↑↑↑	E08	FASLG	17.423
18	↔	A01	ABL1	-1.3157
19	↔	A03	APAF1	-1.1866
20	↔	A05	BAG1	1.0105
21	↔	A08	BAK1	1.4554
22	↔	A10	BCL10	-1.1522
23	↔	B09	NAIP	1.4413
24	↔	B12	XIAP	-1.1584
25	↔	C04	BNIP2	-1.4684
26	↔	C06	BNIP3L	1.0441
27	↔	D04	CASP4	-1.0249
28	↔	D06	CASP6	-1.9333
29	↔	D09	CASP9	1.6979
30	↔	E03	CRADD	1.6262
31	↔	E04	DAPK1	1.436
32	↔	E07	FAS	-1.083
33	↔	E07	FAS	-1.083
34	↔	E10	HRK	-1.5854
40	↓↓	A09	BAX	-7.0745
41	↓↓	A11	BCL2	-9.1946
42	↓↓↓↓	B03	BCL2L11	-17.9496
43	↓	B07	BID	-4.2116
44	↓↓↓↓↓	B08	BIK	-36.5133
45	↓↓↓↓↓	C01	BIRC6	-25.1791
46	↓	C02	BIRC8	-2.1114
47	↓↓	C03	BNIP1	-8.9569
49	↓↓↓↓	C07	BRAF	-18.4811
50	↓	C09	CARD6	-2.7074
51	↓	C10	CARD8	-3.6587
52	↓	C11	CASP1	-2.1089
53	↓↓↓	C12	CASP10	-11.881
54	↓↓↓↓	D08	CASP8	-18.8262
55	↓↓↓↓↓	D11	CD40LG	-47.9471
56	↓	D12	CFLAR	-3.6587
57	↓↓↓	E02	CIDEB	-12.9436
58	↓	E05	DFFA	-3.6587
59	↓	E09	GADD45A	-3.6587
60	↓↓	E11	IGF1R	-9.7576
61	↓	F02	MCL1	-3.6587
62	↓	F03	NOL3	-3.6587
63	↓	F05	RIPK2	-4.5391
64	↓	F09	TNFRSF11B	-3.6587
65	↓↓	F10	TNFRSF1A	-5.2093
66	↓	F11	TNFRSF21	-3.6587
67	↓	F12	TNFRSF25	-3.6587
68	↓	G02	TNFRSF9	-3.6587
69	↓↓↓↓↓	G03	TNFSF10	-23.6357
70	↓	G04	CD70	-3.6587
71	↓	G05	TNFSF8	-3.6587
72	↓	G07	TP53BP2	-3.6587
73	↓	G08	TP73	-4.1904
74	↓	G09	TRADD	-3.6587

*↑, Up-regulation (↑: 2–5, ↑↑: 5–10, ↑↑↑: 10–15, ↑↑↑↑: 15–20, and ↑↑↑↑↑: more than 20-folds); ** ↓, Down-regulation (↓: 2–5, ↓↓: 5–10, ↓↓↓: 10–15, ↓↓↓↓: 15–20, and ↓↓↓↓↓ more than 20-folds); ***↔, No regulation changed

Discussion

Despite extensive research conducted on the treatment of liver cancer to reduce its incidence rate, this type of malignancy is still considerably frequent in the world (Siegel et al., 2014). The present study examined the apoptotic effects of pyrrolidine derivatives on human hepatoma (HepG2) cells. In a similar study, the cytotoxic effects of this derivative were explored on HepG2 cells by the MTT assay, and the cells were double-stained with Annexin-V and PI for the in-vitro flow cytometric detection of apoptotic cells, with the results indicating that it induced HepG2 cell apoptosis significantly at 125µL concentrations after 48 hours (Ramezani et al., 2017). To achieve the research goals, the authors of this article extended the mentioned study making use of the PCR array technology. Apart from developing cancer treatment methods, another way of fighting cancer could be the designing of new therapeutic agents, which act though either the upregulation of pro-apoptotic molecules or the downregulation of anti-apoptotic molecules (Fesik, 2005; Call et al., 2008). In addition, to explore the underlying molecular mechanisms of apoptosis induced by the pyrrolidine derivative, the PCR array technology was used in this article. Research results indicate that pyrrolidine is able to modify apoptosis-induced gene expression (Ramezani et al., 2017). To better understand molecular mechanisms, we specified the expression of apoptosis-related genes by a quantitative real-time PCR array technology and found out that this derivative reduced cell viability and induced apoptosis. This PCR array included a wide range of gene families involved in apoptosis, such as TNF ligands and their receptors, BCL-2 members, caspase, IAP, and TRAF (Table 1). The array data showed that the expression of many genes was connected with the extrinsic and intrinsic apoptosis signaling pathways (Table 2). The perforin-granzyme-dependent killing of the cells was changed significantly together with these changes, with this being remarkably important in the case of anti-apoptotic genes and supporting the assumption that this pyrrolidine derivative was able to exert its apoptotic effects mostly by the downregulation of these genes (Table 1).

As Table 2 shows, the expression of Bcl-2, being known for its anti-apoptotic properties, decreased significantly (Reed, 2008). In the same vein, the expression of other anti-apoptotic molecules, including Bcl2L1, Bcl2L11, IGF1R, BRAF, and CD40LG decreased dramatically. Furthermore, in addition to the two main apoptotic pathways, i.e. the extrinsic and the intrinsic ones, an additional pathway has been detected that includes T-cell mediated cytotoxicity and perforin-granzyme, being dependent on the killing of the cells (Igney and Krammer, 2002). Although the Bcl-2 gene family plays a significant role in both pro-apoptotic and anti-apoptotic pathways, it could be assumed that the reduction in the expression of caspases has been probably due to the fact that these two pathways have been able to affect each other (Table 1) (Reed, 2008). Moreover, the expression of caspases 3 and 5 has increased significantly, with this implying that perforin-granzyme has been further initiated by the cleavage of caspase 3, leading to DNA fragmentation, the degradation of cytoskeletal and nuclear proteins, and the formation of apoptotic bodies (Table 1) (Martinvalet et al., 2005). In the present study, the expression of FASLG l and TNF receptor death domains increased, thereby playing a significant role in signaling pathways, in order of transferring the death signal from the cell surface to the intracellular space (Rubio-Moscardo et al., 2005). In the same vein, P53 played a pivotal role by mediating cellular death after damage, with its pro-apoptotic function having depended on transcription factors, including c-Myc (Table 1) (Marnett and Plastaras, 2001). In the present study, it was demonstrated that the expression of the p53 gene increased significantly, yet another membrane p53 family, i.e. the p73 gene, was downregulated in response to this derivative (Table 2) (Flores et al., 2002).

It is worth noting that the remarkable downregulation of Bcl-2 and IGF1R in the present study could imply that these genes have been the potential targets of cancer drug developments (Chipuk and Green, 2008; Zhou et al., 2017).

In conclusion, as far as the authors of the present article are concerned, these findings are novel and address the apoptotic potential of the new pyrrolidine-2-Carboxamide derivative for the first time. The Pyrrolidine-2-Carboxamide derivative was shown to induce apoptosis in HepG2 cells. It also affected the expression of some genes involved in apoptosis pathways. However, further experiments are required to evaluate these types of compounds to examine their pharmacological properties as the sources of pharmacologically valuable products against human cancer cells.

Funding Statement

Not applicable.