Gallic Acid Anticancer Activity

[Cloris Sopha]

Lungcancer is one of the common malignant tumors,accounting for 12% of all primarily diagnosed cancer cases, and isestablished as the main cause of cancer-associated mortalityworldwide (1,2). Non-small cell lung cancer (NSCLC) is aprominent type of lung cancer, comprising 85% of all lung cancercases worldwide (3,4). Although therapeutic strategiesincluding targeted therapy, immunotherapy and traditionalchemotherapy have achieved considerable success in improving theprognosis of patients with NSCLC over the past decades, themajority of patients still suffer from local aggravation and/orsystemic metastasis, and do not survive >5 years followingdiagnosis (5). Therefore, it isessential to identify novel therapeutics that are capable ofsignificantly elevating the 5-year survival rate while causinglittle side-effects in NSCLC patients.

With advances in research on Traditional ChineseMedicine (TCM), many agents extracted from natural plants haveattracted increasing levels of public attention in recent years fortheir apparent favorable pharmacokinetic characteristics and mildside-effects. Gallic acid (3,4,5-trihydroxybenzoic acid; GA), anatural phenolic compound, is one such plant extract that ispresent in abundance in tea, grapes, gall-nuts and red wine(6,7). It has been reported to possess variouspharmacological and biological properties, including antibacterial,antiviral and antitumor activities. Recently, there has been anincreased research focus on the antitumor capacity of GA indifferent cancer cell lines, including oral, lung, pancreatic andcervical cancer cells (8,9), and it is thought that regulation ofapoptosis may be critically involved in the antitumor effects ofGA. However, understanding of how GA induces cell apoptosis isstill limited.

Taking these findings into consideration, thepresent study hypothesized and investigated whether GA exerts itsanticancer effects on NSCLC A549 cells by modulating thephosphorylation of JAK1 and STAT3, and the expression of downstreamapoptotic molecules, including B-cell lymphoma 2 (Bcl-2) andBcl-2-associated X protein (Bax). Notably, it was also evaluatedand confirmed that GA facilitates the anticancer effects ofcisplatin in A549 cells by regulating the JAK/STAT3 signalingpathway.

The A549 cell line was maintained in RPMI-1640medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/mlstreptomycin, in a humidified incubator at 37C with 5%CO2. Cells at logarithmic phase were used in thefollowing experiments.

Cells were cultured in 24-well plates on sterileglass coverslips placed in each well and treated as aforementioned.The cells were then fixed at 4C with 4% paraformaldehyde for 30min and washed twice with PBS. Triton X-100 solution (0.1%) wasused to disrupt the cytomembrane, then cells were blocked in 10%normal goat serum (Beijing Solarbio Science & Technology Co.,Ltd.) at room temperature for 1 h, and incubated with p-STAT3(dilution, 1:1,000; cat. no. P00007) at 4C overnight.Subsequently, the cells were incubated with biotin-labeledsecondary antibody (dilution, 1:64; cat. no. BA1090; Wuhan BosterBiological Technology Co., Ltd.) for 40 min at 37C. Finally,nuclei were stained with DAPI at room temperature for 1 min and theslides were observed with a fluorescent microscope (OlympusCorporation; magnification, 200).

All quantitative data were presented as the mean standard deviation, and statistical analysis was performed withSPSS 19.0 (IBM Corp., Armonk, NY, USA). One-way analysis ofvariance followed by the Least Significant Difference post hoc testwas applied to analyze the differences among groups. P

To investigate the influence of GA on apoptosis,A549 cells were treated with 12, 20 or 28 g/ml GA for 24 h and thenumber of apoptotic cells was calculated by flow cytometry. Theresults demonstrated that 12 g/ml GA significantly increased thepercentage of early and total apoptotic cells when compared withthe control group following 24 h of incubation (P

To further ascertain the antitumor effects of GA inA549 cells, the expression of Bax and Bcl-2 in cells from eachgroup was measured by RT-qPCR and western blot analysis. Theresults revealed that GA upregulated Bax and downregulated Bcl-2 atthe gene and protein levels (Fig.3). Specifically, 12 g/ml GA enhanced Bax protein (Fig. 3A) and gene (Fig. 3C) expression relative to the levelsin control cells (P

It is widely accepted that the JAK/STAT3 signalingpathway is involved in various biological processes, including cellproliferation, survival and development (14). To determine whether the JAK/STAT3signaling pathway was associated with the anticancer effects of GA,the expression of JAK1 and STAT3 was examined in A549 cells fromeach group by RT-qPCR and western blotting. The results revealedthat 12 g/ml GA reduced the levels of p-JAK1Y1022 andp-STAT3Tyr705 when compared with control treatment(P

To confirm whether GA influenced the stimulatoryeffects of cisplatin on apoptosis, A549 cells were treated with GA,cisplatin or the two combined for 6 or 24 h, and cell apoptosis wasmeasured by cytometry. The results indicated that cisplatintreatment for 6 or 24 h increased the apoptosis of A549 cells byvarying extents (P

The morphological changes of cells from differentgroups were observed by crystal violet staining assay (Fig. 6). Cellular structure was intact inthe control group, while cells exhibited apparent apoptotic changessuch as cell shrinkage, nuclear chromatin condensation andfragmentation when treated with GA, cisplatin, or a combination ofthe two agents for 24 h. Additionally, cells in the experimentalgroups exhibited an evident decrease in the number of cells;notably, combined treatment with GA and cisplatin together lead tothe greatest decrease in cell number. These results revealed thatGA or cisplatin treatment alone resulted in morphological changesin A549 cells, though their combination could lead to more evidentchanges.

To determine whether GA could enhance the effects ofcisplatin on the regulation of the JAK/STAT3 signaling pathway,several major molecules associated with apoptosis, anti-apoptosisand proliferation in cells were examined by western blot andimmunofluorescent staining assays. The results demonstrated that GAor cisplatin treatment alone increased Bax protein expression anddecreased Bcl-2 protein expression (P

The expression of the JAK/STAT3 signaling pathway incells treated with GA, cisplatin or the two agents combined wasalso evaluated. The results demonstrated that the levels ofp-STAT3Tyr705 and p-JAK1Y1022 weresignificantly decreased in cells treated with GA or cisplatin alone(P

Lung cancer is one of the most common types ofcancers and is characterized by a high mortality rate andresistance to chemo- and/or radiation therapy is easily acquired(16). For the majority of patientswith NSCLC, there is difficulty in selecting the optimumtherapeutic regimens. Cisplatin-based chemotherapy has achievedconsiderable success in improving the prognosis and 5-year survivalrate of patients compared with non-cisplatin regimens. However, theuse of cisplatin is markedly limited by its side-effects, includingnephrotoxicity, severe nausea and vomiting (17). There is an urgent requirement toidentify novel drugs with little or no side-effects. Recently,plant-derived compounds have attracted increasing levels of publicattention for their potential anticancer activities and lowtoxicity. GA is one such product that exists in various plants andmay possess anticancer activity in various cancer cells includingthose of lung cancers (8). Aprevious study has reported that GA could enhance the effects ofchemotherapeutic agents in lung cancer (7). However, the underlying mechanisms arestill not fully understood.

It is well known that apoptosis is a strictlyprogrammed cell death process, which serves a critical role inmaintaining the balance between cell survival and death (18). Normally, apoptosis is a criticallyregulated physiological process; however, abnormal cellularproliferation and accumulation of genetic defects may occur ininstances of impaired apoptotic mechanisms, which could furtherlead to tumorigenesis and resistance to treatment (19). Therefore, abnormal cellularproliferation and evasion of apoptosis are considered to behallmarks of cancer, and the majority of antitumor drugs exerttheir effects by inhibiting cellular proliferation and inducingcell apoptosis. In the present study, the anticancer capacity of GAand its auxiliary effects on cisplatin were evaluated, and theresults demonstrated that GA and cisplatin had marked effects ondecreasing A549 cell viability in dose- and time-dependent manners.Notably, combined treatment with GA significantly enhanced theeffects of cisplatin. The present study has also identified thatindividual GA or cisplatin treatment induced apoptosis in A549cells, and furthermore, cotreatment with GA enhanced theapoptosis-inducing effects of cisplatin. These results wereconsistent with previous studies reporting that GA inhibited thegrowth and induced the apoptosis of hepatic stellate (6), prostate cancer (8) and ovarian cancer cells (9).

Apoptotic pathways are known for their functions inmodulating the balance between cell proliferation and apoptosis byregulating the expression of a series of growth factors, cytokinesand vasoactive substances (20). Animbalance between cell proliferation and apoptosis is one of themain causes of tumorigenesis (21).Among the key factors involved, the JAK/STAT3 signaling pathway hasrecently gained increased research focus. Transient activation ofthe JAK/STAT3 signaling pathway in normal tissue is involved innumerous fundamental biological processes, including cellproliferation and apoptosis, and the development of organs(22). However, persistentactivation of the JAK/STAT3 signaling pathway has been observed inseveral types of cancers including NSCLC (14). Inhibition of the JAK/STAT3 signalingpathway has therefore been recognized as a promising therapeuticstrategy for NSCLC. In addition, the JAK/STAT3 pathway may regulatemany gene products associated with apoptosis and anti-apoptosis,including Bax and Bcl-2 (23).

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