The Effects of Aspirin on Bax, Bcl-2 and iNOS Expression Level in Hela Cancer Cell Line

 Cervical cancer develops in female cervix. 99% of cervical cancer cases are linked to infection with high-risk human papillomaviruses (HPV). Cervical cancer is the fourth most common cancer in women. Effective primary (HPV vaccination) and secondary prevention approaches can prevent most cervical cancer cases. The treatment of cervical cancer varies worldwide. Radiation may be used in all stages where surgical options do not exist. In addition, chemotherapy can be used to treat cervical cancer, and has been found to be more effective than radiation alone. However, prevention methods are very important to be considered to overcome the soaring incidence of cervical cancers (1,2). Nonsteroidal anti-inflammatory agents (NSAIDs) are a group of medicines that relieve pain and fever and reduce inflammation. They work by blocking a specific group of enzymes called cyclo-oxygenase enzymes (COX enzymes) leading to reducing of prostaglandins production. Aspirin, also known as acetylsalicylic acid (ASA), is an NSAID used to reduce pain, fever, or inflammation. Aspirin is also used long-term to help prevent further heart attacks, ischaemic strokes, and blood clots in people at high risk. Aspirin decomposes rapidly in some chemical solutions including ammonium acetate or the acetates. Aspirin's ability to suppress the production of prostaglandins and thromboxanes is due to its irreversible inactivation of the cyclooxygenase enzyme required for prostaglandin synthesis. Aspirin also uncouples oxidative phosphorylation in certain tissues mitochondria, by diffusing from the inner membrane space as a proton carrier back into the mitochondrial matrix, where it ionizes once again to release protons. There is evidence that has shown that aspirin as a chemoprotective agent may reduce overall cancer incidence and mortality in colorectal, esophageal and gastric cancers with smaller effects on prostate, breast and lung cancer. Research has shown that NSAIDs such as aspirin play a role in preventing cancer development in a variety of organs including colon, pancreas, stomach, uterus and esophagus (3,4). In vitro and in vivo studies have revealed that NSAIDs have significant inhibitory effects on a variety of tumors, including gastrointestinal tumors and tumors of the reproductive system. Recent research suggest that NSAIDs have inhibitory effects on ovarian, breast, and cervical cancer cells in vivo and in vitro (5-10).  Aspirin has been reported to have antitumor effects on reproductive cancer cells (11). However, contrary to research findings that confirm the anti-tumor effects of aspirin on cancer cells of the reproductive system, some research has shown that aspirin has no significant effects on cancer development. Some research results have not shown a significant association between aspirin and reduced endometrial and ovarian cancers (12, 13). Although many studies demonstrate the anti-cancer effects of aspirin, the inhibitory effect of aspirin on cancer cells are still challenging. The present study investigated the cytotoxic effects of aspirin on cervical cancer (Hela) cells in vitro and the effects of cytotoxic concentration of aspirin on expression level of apoptotic BAX, anti-apoptotic Bcl-2 and iNOS in cervical cancer cells.

In this experimental-laboratory study, cervical cancer cell line was purchased from Pasteur Institute and divided into aspirin-treated groups with 0.0001, 0.001, 0.01,0.1, 1 and 10 mg /ml, and control (untreated) group. MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) assay was used to determine cell viability through determination of mitochondrial function of cells by measuring activity of mitochondrial succinate dehydrogenase, during which, MTT is reduced to a purple formazan by NADH. The product was quantified by light absorbance at 570 nm. The expression levels of BAX, Bcl-2, iNOS genes were evaluated by Reverse transcription-polymerase chain reaction (RT-qPCR) technique. Total RNAs were extracted with the high purity RNA extraction kit according to the manufacturer’s instructions and reverse-transcribed into cDNAs. Then, real-time quantitative PCR was conducted to analyze Bax , Bcl-2, iNOS and GAPDH expression levels. The expression of genes was calculated based on 2-ΔΔCT method and was normalized to the loading control, GAPDH. Data were analyzed using one-way ANOVA.

The results of the present study showed that cervical cancer (Hela) cell viability did not change significantly when exposed to 0.0001, 0.001, 0.01 and 0.1 of aspirin compared to control group. However, exposure of cervical cancer cells to 1 and 10 mg / ml of aspirin significantly reduced cell viability (p<0.05 and p<0.001, respectively). BAX, Bcl-2 and iNOS expression levels significantly increased in cervical cancer cells exposed to1 mg/ml of aspirin. The BAX / Bcl-2 ratio was 3.18 showing the higher level of apoptotic BAX than anti-apoptotic Bcl-2 expression level.

The results of this study showed that lower concentrations of aspirin did not have cytotoxic effects on cervical cancer cells, but higher concentrations could induce BAX-dependent apoptosis by increasing the ratio of BAX to Bcl-2 expression level, and increasing the relative expression of Nitric oxide synthase gene. After binding to its receptor and triggering a chain of reactions, aspirin is thought to trigger the expression of apoptotic genes by acting on DNA and transcription. In this way, BAX in the mitochondrial membrane, after homodimerization and oligomerization, causes the opening of anion channels, and consequently the potential difference of the mitochondrial membrane changes. These channels release proteins and apoptotic factors such as cytochrome C into the cellular cytosol, and following this release, apoptosis occurs with the activation of caspase cascade. In this study, the cytotoxic concentration of aspirin, in addition to significantly increasing the expression of BAX apoptotic gene, unexpectedly increased Bcl-2 anti-apoptotic gene, but due to the very high ratio of BAX to Bcl-2, increased gene expression of anti-apoptotic Bcl-2 was not able to counteract the high level of BAX expression and thus the process of induction of apoptosis has occurred in cervical cancer cells. The results of this study showed that aspirin increases the expression level of iNOS gene, which in turn plays a role in apoptosis induction in cervical cancer cells. Conclusively, lower concentrations of aspirin (and physio-pharmacological doses) do not have significant anticancer effects on cervical cancer cells. However, further experimental, clinical and epidemiological studies are required to determine the exact anticancer effects of aspirin on the cervical cancer cells.