Effects of Resveratrol against Lung Cancer: In Vitro and In Vivo Studies

Uncontrolled cell growth and resistance to apoptosis characterize cancer cells. These two main features are initiated in cancer cells through mutations in key signaling molecules, which regulate pathways that are directly involved in controlling cell proliferation and apoptosis. Resveratrol (RSV), a naturally occurring plant polyphenol, has been shown to have biological effects counteracting different diseases. It has been found to provide cardio-protective, neuro-protective, immuno-modulatory, and anti-cancer health benefits. RSV has been found to inhibit cancer cell proliferation, induce cell cycle arrest and apoptosis, and these anticancer effects may be due to its ability to modulate signaling molecules involved in these processes. The present review summarizes the existing in vitro and in vivo studies on resveratrol and its anti-lung cancer properties.


Introduction
Cancer cells are most commonly identified by their high rate of proliferation and resistance to programmed cell death/apoptosis [1]. Cancer cells are self-sufficient in growth signals, and insensitive to anti-growth signals. Their cell replication is up regulated while their programmed cell-death pathway (apoptosis) is down regulated. They have sustained angiogenesis, ensuring that the cells are vascularized by blood vessels and they have an ability to invade tissues and metastasize [1].
Lung cancer is a malignant lung tumor characterized by uncontrolled cell growth in lung tissue. It is the most common cancer among men in both incidence and mortality. Among women, lung cancer is the third highest in incidence and second in mortality after breast cancer. In 2012, there were 1.82 million new cases and 1.56 million deaths globally due to lung cancer, representing approximately 19.4% of all deaths from all types of cancer [2].
Plant derived compounds and bioactive food components have recently emerged as agents with anticancer properties [3][4][5][6]. One such bio-molecule is resveratrol (trans-3,5,4 -trihydroxystilbene) [3]. Resveratrol (RSV) is a naturally occurring polyphenol that is most commonly found in grapes, red wine, and peanuts [7]. Specifically, grape skin contains 50-100 mg of RSV per gram [8] but the dominant source of RSV is the root of Polygonum cuspidatum, which is widely used in traditional Chinese and Japanese medicine [9,10]. The RSV concentration in red wine is significantly higher than the respective concentration in white wine, a fact that is attributed to the differences in the wine fermentation processes. The average concentration or RSV in red wine ranges from 0.84 to 7.33 mg/L [11], while, in white wine, ranges from 0.01 to 0.08 mg/L [12,13].
Resveratrol has been found to have health benefits such as protection against cardiovascular disease, aging, metabolic disease and cancer [7]. The most known property of RSV is its antioxidant effect which allows it to convert free radical like reactive oxygen species (ROS) to unreactive compounds [14]. A groundbreaking study by Jang et al. found that resveratrol has anticancer growth inhibition were studied. It was found that SA-β-gal expression was increased, p53 and p21 expression was increased, double-stranded DNA breaks were increased, ROS levels were elevated, Nox5 expression was up-regulated, and EF1A expression was decreased [36]. A549 cells pre-treated with Benzo(a)pyrene followed by RSV treatment, resulted in a decrease in cell viability, an increase in p53 levels, G2/M cell cycle arrest and an induction of apoptosis. Bcl-2 down-regulation, decreased cyclin D expression, down-regulated NF-κB and Iκκ1 expression, increased p21 expression and increased TRAIL receptors 1 and 2 expression [37]. A549 cells treated with RSV showed decreased cell proliferation, inhibition of TGF-β1 induced epithelial to mesenchymal transition (EMT) and suppression of cell adhesion [26]. Dose-dependent cell growth inhibition and activation of caspase-3 of A549 cells was observed with RSV treatment [38]. RSV treatment of CL-1-5, A549, H322 and H1435 cells resulted in inhibition of growth/proliferation, down-regulated Akt, IκB and NF-κB [39]. TMS (an analog of RSV) treatment of H1975 cells increased intracellular calcium levels in Gefitinib resistant NSCLC. TMS treatment was found to only induce anti-proliferative effects in Gefitinib resistant NSCLC, not normal NSCLC and normal lung epithelial cells. This was associated with decreased EGFR phosphorylation and activation, induction of caspase-independent apoptosis and autophagy by direct binding to SERCA and inducing ER stress and AMPK activation, suppression of the mTOR pathway and increased JNK activity [40]. Lucas et al. showed that A549 cells treated with RSV reduced growth mediated through an induction of caspase-3 activity [41]. Treatment with RSV conjugated nanoparticles (NP) followed by cigarette smoke condensate (CSC) showed that RSV alone attenuated CSC-induced DNA fragmentation in H727 cells. However, NPs dramatically increased RSV induced apoptosis in CSC-treated cells. These results indicate that NPs are capable of increasing the efficacy of lipophilic drugs such as RSV [42]. A549 cells treated with RSV exhibited a decrease in cell proliferation, cell viability and cell cycle arrest that was associated with reduced AK001796 non-coding RNA [43]. A549 cells treated with RSV showed reduced proliferation as well as G0/G1 cell cycle arrest, increased in p53 nuclear expression, down-regulation of cyclin D1, CDK4 and CDK6 expression as well as an up-regulation of the CDK inhibitors, p21 and p27 [44]. A549 cells treated with RSV showed reduced P62 levels, increased p62 degradation and an increase in Fas/Cav1 complex formation [20]. Fas/Cav1 activated complexes lead to an increase in caspase-8 mediated Beclin-1 cleavage, resulting in c-terminal Beclin-1 translocation to the mitochondria to initiate apoptosis [32]. A549 cells treated with THS (an analog of RSV) showed a dose-dependent induction of apoptosis and autophagy. Up-regulation of cleaved PARP, increased caspase-3 and -9, increased LC3-II accumulation, down-regulation of Bcl-2, inhibition of the mTOR pathway and elevated ROS levels were also observed in these cells [45]. A549 cells treated with RSV-loaded nanoparticles had reduced H 2 O 2 induced ROS levels, increased RSV uptake, activated Nrf2-Keap1 signaling and accumulation of Nrf2 [46]. A significant inhibition of LLC cell growth was seen with treatment with DHS (an analog of RSV). It was also accompanied by a decrease in LLC cell migration and invasion [47]. CEM and A549 cells treated with RSV showed a decrease in proliferation, an increase in apoptosis, an inhibition of tubulin polymerization, G2/M cell cycle arrest, loss of the mitochondrial membrane potential as well as activation of caspase-3 and -9 and PARP cleavage [48].

Effects of Resveratrol (RSV) in a Combination Treatment against Lung Cancer: In Vitro Studies
There have also been several in vitro studies looking at the effect of RSV as part of a combination treatment (Table 2). These studies are summarized in the following section. A549 and H460 cells pre-treated with RSV followed by Gy IR treatment resulted in a synergistic enhancement of the IR-induced cell killing in NSCLC through an apoptosis-independent mechanism. This apoptosis-independent mechanism was found to be increased percentage of SA-β-gal positive senescent cells and an increase in double-stranded DNA breaks [52]. H-2452 cells treated with a combination of RSV and Clofarabine showed a synergistic decrease in Msl-1 protein expression with little effect on Bcl-xL expression [41]. However, it was found that Bcl-xL knockout enhanced the ability of the combination treatment to inhibit cell proliferation and increase apoptosis [41]. The increase in apoptosis found through the combination treatment of these cells was found to be mediated through G2/M phase cell cycle arrest and increased caspase-3 and -7 activity as well as an increase in caspase-3 cleavage [53]. H460, A549, PC-9 and H1975 cell treatment with a combination of RSV and Erlotinib resulted in a synergistic enhancement of Erlotinib-induced apoptosis, reduced cell viability and colony formation [42]. An increase in ROS production, reduction in expression of anti-apoptotic proteins, such as survivin and Mcl-1, promotion of p53 and PUMA expression, increased caspase-3 activity was observed and the combination was more effective at inhibiting the Akt/mTOR/p70s6K pathway [54]. A549 cells grown as spheroid bodies to resemble cancer stem cells (CSCs) were transfected with the ZD55 oncolytic adenovirus and treated with RSV in a study by Yang et al. [39]. The ZD55 virus was carrying the TRAIL gene (ZD55-TRAIL). ZD55-TRAIL alone induced cytotoxicity, however, the combination treatment increased the ZD55-TRAIL mediated cytotoxicity [39]. It was also found that the induction of apoptosis in these cells was caspase dependent with decreased levels of pro-caspase-9, -8 and -3 [51]. Zhu et al. showed that PC9/G treatment with Gefitinib and RSV resulted in a synergistic inhibition of proliferation of Gefitinib-resistant NSCLC cells [43]. Co-treatment also induced apoptosis, autophagy, cell cycle arrest and senescence [43]. These effects were found to be mediated through an inhibition of EGFR phosphorylation by increasing Gefitinib accumulation intracellularly, increased CYP1A1 and ABCG2 expression and increased expression of cleaved caspase-3, LC3B-II, p53 and p21 [55]. Finally, Lung fibroblasts affected with idiopathic pulmonary fibrosis pre-treated with RSV followed by TGF-β or CXCL12 showed that RSV is capable of repressing fibroblast to myofibroblast conversion in lung cells. It was also shown that RSV is capable of reversing fibroblast to myofibroblast conversion in lung cells if they were treated with TGF-β and CXCL12 followed by RSV [56]. Fibroblast to myofibroblast conversion is reversed and repressed in lung and prostate fibroblasts (1) RSV 50 µM and below repressed and reversed myofibroblast phenoconversion, but had no effect on N1 or primary prostate fibroblast cell proliferation, apoptosis or COL1 and EGR1 gene transcription.
(2) RSV 100 µM and above induced the same effects observed in N1 and primary prostate fibroblast, in IPF lung fibroblasts

Effects of Resveratrol (RSV) in Lung Cancer: In Vivo Studies
Few studies have looked at the effect of resveratrol treatment of lung cancer in vivo (Table 3). Female nude mice (five weeks old) injected subcutaneously with A549 cells were treated with 50 mg/kg DHS, an analog of resveratrol, intraperitoneally from Day 1 to Day 4 and then Day 7 to Day 10. This treatment regimen resulted in a decrease in tumor growth [26]. Zhao et al. reported that 18 female Balb/c mice injected subcutaneously with SPC-A-1 cells into their flanks showed an inhibition of tumor growth upon treatment with 1 or 3 g/kg/day RSV for 28 days in their diet [27]. Nude mice inoculated with A549 cells were treated with 20 mg/kg RSV every other day for 25 days, resulted in an inhibition of metastasis and activation of SIRT1 [57]. Nude mice subcutaneously injected with A549 cells were treated with 15, 30 or 60 mg/kg RSV injections for 15 days in a study by Yin et al., which found that RSV inhibits lung cancer growth in a dose-dependent manner [38]. Male mice given 100 mg/kg body weight Benzo(a)pyrene (BP) to induce lung carcinogenesis were treated with 5.7 µg/mL RSV in drinking water and 60 mg/kg body weight curcumin for 22 weeks. The BP-treated mice showed increased levels of p53 hyper-phosphorylation and decreased activity of caspase-3 and -9. The combination treatment lead to an increase in caspase-3 and -9 activity and reduced p53 hyper-phosphorylation, which completely counteracted the activity of BP treatment [58]. Finally, Savio et al., studied the effect of 25 mg/kg/day DHS in drinking water for seven days on sixty male C57B6 mice (four weeks old) bearing LLC tumors, and a decrease in tumor volume, cell proliferation, tumor angiogenesis and liver metastatic lesions was observed [47]. LLC tumor cells were injected subcutaneously into the flanks of C57BL/6 mice and treated with RSV from Day 10 after the injection until their sacrifice (four weeks after injection). RSV treatment led to an inhibition of lung cancer tumor growth through reduction of F4/80+ expressing cells and M2 polarization in tumors [59]. RSV (Resveratrol); DHS (Dehydrosilybin); LLC (Lewis lung carcinoma); SIRT1 (silent mating type information regulation 2 homolog 1); BALB/c (Bagg Albino/c); BP (Blood pressure); ↑ (increase); ↓ (decrease).

Molecular Targets/Signaling Molecules Contributing to Anticancer Effects of RSV
Cancer cells overexpress growth factor receptors, such as the epidermal growth factor receptor (EGFR) [60] and glucose transporters [61] giving them advantages towards proliferation and survival. Binding of growth factors to their respective receptors enhances the intrinsic tyrosine kinase activity of the receptor and activates signaling pathways, including the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, that leads to increased proliferation and inhibition of apoptosis [62]. Activation of the growth factor receptors also leads to activation of the Ras-mitogen activated protein kinase (Ras-MAPK) cascade, resulting in enhanced proliferation [63]. On the other hand, the pathway of p53, a well-established tumor suppressor, is often found down-regulated, if not silenced, in many types of cancer cells [50].

Conclusions
Cancer is defined by a high rate of mutation often inducing chemotherapeutic resistance to established treatments and, in recent years, there has been an increasing focus on finding new cancer therapies. From the studies summarized in this review, it is evident that resveratrol is capable of: (1) decreasing cancer cell proliferation and inhibiting tumor growth; (2) inducing cell cycle arrest; (3) inducing cell apoptosis; and (4) inhibiting metastasis of lung cancer. In many studies, RSV was found to enhance ROS production in cancer cells inducing cytotoxicity.
Resveratrol has low bioavailability and the plasma resveratrol concentrations have been reported to be at nanomolar or lower levels. Most of the studies presented here used concentrations in the µM range, which may not accurately represent physiologically relevant concentrations; however, one study showed that resveratrol conjugated NPs increased the activity of resveratrol and enhanced its uptake by cells. This poses a promising and novel area of research to help increase uptake of lipophilic drugs such as resveratrol, which in general tend to have very low bioavailability. Further research should be conducted on resveratrol bioavailability and resveratrol metabolites to understand the mechanism behind the resveratrol induced anti-cancer effects. Resveratrol was also shown through numerous studies presented here to not only act synergistically with chemotherapeutic drugs to increase their anti-cancer effects, but it was also shown to reverse resistance to those chemotherapeutics, which is very desirable as the prevalence of chemotherapy resistant cancer cells increases. All together, these findings present resveratrol as a promising anti-cancer treatment and warrant further studies to better understand the mechanism behind resveratrol induced anti-cancer effects.