Vanadium (IV) is a metal ion complex which has been introduced as a candidate to treat the cancers (Nair et al., 2014). Due to the lowest side effects of Vanadium IV, when compared to other metal ions such as platinum (Leon et al., 2017a), current studies are focused on the complexes to introduce a safe effective drug. Our previous investigation revealed that the IV complex with 4-bromo-2-(((5-chloro-2-hydroxyphenyl) imino) methyl) phenol (L), which abbreviate to [IV(L)] complex, induces apoptosis in the HepG2 cell lines higher than L929, as a normal cell line (Aliabad et al., 2018).It has been reported that IV complexes may overcome cancer cells vi several mechanisms including up-regulation of free radical reactions (Wang et al., 2010), altered expression in the molecules involved in the apoptosis, such as phosphp21 activated protein kinases (PAK), oinositide-3-kinase–protein kinase B/Akt (PI3K-PKB/Akt), cyclin-dependent kinase (CDK) 4, 6 and 7, death-associated protein kinase (DAPK), protein 2-alpha (AP2), Fas-associated protein with death domain (FADD), c-Jun N-terminal kinase (JNK), Caspase3 (CASP3), CASP6, CASP7, CASP10, CASP11 and B-cell lymphoma-extra (Bcl-x), (Leon et al., 2017b, Leon et al., 2016). However, the effectiveness of the [IV(L)] complex on the expression of P53, caspase-8, Bax, Bcl-2, Bim, P21 and Bid molecules in the HepG2 and L929 cell lines is yet to be clarified. Therefore, based on our previous investigation which confirm the more cytotoxicity of [IV(L)] complex on the HepG2 than L929 cell lines, the main aim of this study was to find the main molecules which are the target of [IV(L)] complex to induce apoptosis in the cancerous cell line.
Materials and Methods
Cell lines and cell culture conditions
HepG2, the human liver cancerous cells, and mouse fibroblast L929 cell lines were purchased from Pasture Institute, Tehran-Iran. The cell lines were cultured in the RPMI 1640 culture medium in a standard condition have been described in our previous investigation (Aliabad et al., 2018). Determination of inhibiting cell growth by 50% (IC50), percent of survived cells by using MTT assay (Roche CO. Mannheim, Germany), preparation of 4-bromo-2-(((5-chloro-2-hydroxyphenyl)imino)methyl)phenol (H2L) and 2,2′-bipyridine)[4-bromo-2-((5-chloro-2- hydroxyphenylimino)methyl)phenol] oxido-vanadium(IV) [VOL(bipy)] as well as FTIR spectral data, were described in details in our previous investigation (Aliabad et al., 2018).
RNA extraction and cDNA synthesize
Total RNA was purified from HepG2 and L929 cell lines before and 48 hours after treatment with [IV(L)] complex using RNX solution from Cinnaclon Company, Tehran, Iran. cDNA also were synthesized using a commercial kits from Parstous Company, Mashhad, Iran. Briefly, total mRNA, oligo-dT and RNase free water were added to the tubes and incubated at 70ᵒC and then 4ᵒC for 10 and 4 minutes, respectively. Then, the premix was added and incubated at 40ᵒC for 60 minutes.
Real-time PCR was performed using a master mix from Parstous Company, Mashhad, Iran, and specific primers for P53, caspase-8, Bax, Bcl-2, Bim, p21 and Bid (Table 1). Real-Time PCR protocol was run in a Real-Time PCR machine, Bio-Rad CFX96, as follow: start hot temperature by 95ᵒC for 5 minutes following with 45 cycles of 95ᵒC (30 Seconds), 58ᵒC (30 Seconds) and 72ᵒC (30 Seconds). Β-actin was used as housekeeping gene and the raw data was calculated using 2-∆∆ct.
After evaluation of the normality distribution of the data, One Way ANOVA under SPSS software version 18 was used to analyze the data.
Data analysis revealed that p53 mRNA levels were significantly increased in both HepG2 (3.04 ± 1.39 fold changes, p 0.05). Bcl-2 mRNA levels were decreased 3 folds in the HepG2 cell lines (p 0.05), the values were significantly increased in the L929 cell lines (p
The results also demonstrated that mRNA levels of P21 were increased significantly in either HepG2 (p 0.05) when compared to the treated cell.
It has been reported that HCC is a prevalent cancer among developed and developing countries (Winters et al., 2017, Tervonen et al., 2017). Although HCC is a drug resistance in some cases (Galun et al., 2017, Dong et al., 2017, Guo et al., 2014), our previous investigation revealed that [IV(L)] complex treatment led to significant apoptosis in HepG2 cell lines when compared to L929, the normal liver cell line (Aliabad et al., 2018). However, the main mechanisms leads to apoptosis in the HepG2 cell line in dependent of [IV(L)] complex are yet to be clarified. Thus, the current study was designed to explore the effects of [IV(L)] complex treatment on the expression of the pro-apoptotic molecules including P53, caspase-8, Bax, Bcl-2, Bim, P21 and Bid.
The results demonstrated that all of the pro-apoptotic molecules were significantly increased in the L929 cell line, except Bax which was down-regulated after treatment with [IV(L)] complex. Although HepG2 cell line showed similar patterns of the expression of the molecules, except Bid and caspase-8 which were down-regulated, the effects of [IV(L)] complex on the expression of the pro-apoptotic molecules were not as well as its effects on the L929 cell line (Table 1). Thus, it may be concluded that [IV(L)] complex induces apoptosis in the It has been demonstrated that L929 cell line is more sensitive to [IV(L)] complex than HepG2 cell line. This is in parallel with our previous investigation which revealed that [IV(L)] complex IC50 is lower for L929 than HepG2 cell lines (Aliabad et al., 2018). However, our previous investigations revealed that early apoptosis were significantly higher in the HepG2 than L929 cell lines (Aliabad et al., 2018).
Due to the fact that normal cells have a normal positive feedback of the intracellular signaling pathways, hence, it may be hypothesized that although mRNA levels of the pro-apoptotic molecules were increased in the normal cell, positive feedback of anti-apoptotic molecules may regulate the effects of [IV(L)] complex and inhibit the apoptosis. Interestingly, although the pro-apoptotic molecules were not increased in the HepG2 as L929 cell line, it appears that asymmetrical expression of pro and anti-apoptotic molecules in the cancerous cells, here HepG2, led to more apoptosis in the HepG2 than L929 cell lines. Interestingly, Bax decreased in both cell lines. Due to the fact that Bax gene expression is regulated by P53 and P53 induces apoptosis via several mechanisms, including up-regulation of Bax, thus it may be demonstrated that [IV(L)] complex induces apoptosis in both cell lines independent of Bax molecule. And due to the significantly increased expression of P53, the molecule uses other mechanisms rather than Bax to induce apoptosis.
Additionally, no alteration in the expression of Bim and also down-regulation of Bid in the HepG2 cell line, propose that the molecules also do not involve in the induction of apoptosis in the HCC cell line. Nair and colleagues showed that nicotinoyl hydrazones component of Vanadium up-regulates P53 in the SiHa and HeLa cancerous cell lines and subsequently increased apoptosis in the cell lines (Nair et al., 2014). Thus, it appears that P53 is a main target of [IV(L)] complex to up-regulate and induction of apoptosis in the HepG2 cell line. Additionally, the results demonstrated that P21 had the most increased expression among the pro-apoptotic molecules. Interestingly, Zhang et al., reported that vanadium induces apoptosis in the C141 tumor cell line by up-regulation of P21 in P53 dependent manner (Zhang et al., 2002). Additionally, due to the results regarding the up-regulation of caspase-8 in the HepG2 cell line, and based on the roles played by caspase-8 in the external pathway of apoptosis, it appears that [IV(L)] complex may also target the cells significantly via the pathway.
Taken together, [IV(L)] complex is an important chemical agents which induces apoptosis in the HepG2 cell line by increasing in the expression of two important pro-apoptotic molecules, P53 and P21, and external apoptosis pathway. Moreover, it has been documented that Sodium orthovanadate, another component of Vanadium, inhibits human HCC cells by increased autophagy (Wu et al., 2014). Due to the fact that autophagy is a mechanism, which is different from apoptosis therefore, it may be hypothesized that [IV(L)] complex may suppress HepG2 cell line growing by a combination of apoptosis and autophagy which needs to be explored by further investigations. Moreover, it appears that normal cells also are the targets of [IV(L)] complex to be killed, hence, using the complementary protective materials can reduce the side effects. Wang et al., reported that antioxidants shows synergistic effects when used in association with Vanadium to reduce their toxicities on human normal cells (Wang et al., 2010). Thus, it may be proposed that using antioxidants may decline the L929 sensitivity to the [IV(L)] complex.