| Cancer, next only to heart disease, is the second leading cause of death in developing countries. The burden of cancer is increasing in economically developing countries as a result of environment pollution,population aging and growth as well as, increasingly, an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, and''westernized"diets. Cancer can be treated by surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy or other methods.Chemotherapy plays a main role in the anti-tumor therapy. Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. Ideally, chemotherapeutic agents should target cancer cells specifically without affecting normal cells, by inducing cytostatic or cytotoxic effects. In fact, the lack of specificity, intrinsic or acquired drug resistance, especially the induction of side effects due to high dosage affects the therapeutic effect of many chemotherapeutics. In addition to finding more effective substances of low toxicity, these problems can also be overcome by combining other compounds or drugs with a lower dose treatment agents to get an additive or synergistic therapeutic effect. Using the cell cycle-specific agent to interfere the cell cycle and block the cancer cells at a certain period, will improve the sensitivity of cancer cells to chemotherapeutics. Meanwhlie,it can also decrease side effect by reducing the dose of chemotherapeutics.Among the chemotherapy drugs, Fluorouracil (5-Fu) is widely used in the treatment of a range of solid tumors as well as for cutaneous diseases. It works by blocking the cancer cells at G1/S phase and induces apoptosis of cancer cells by inhibiting essential biosynthetic processes, or by being incorporated into RNA and DNA as a thymidylate synthase inhibitor to influence their normal function. In the process, 5-Fu also destroys normal cells, causing several side effects. In addition, 5-Fu resistance during the course of treatment has become common nowadays, which is an important cause of failure for cancer therapies. To reduce the adverse effects and maximize the anti-tumor effects of 5-Fu, efforts have been made in combination of 5-Fu with several second-line agents, such as paclitaxel, mitomycin, leucovorin, tegafur, and so on. In addition, some natural phytochemicals have also shown increased effects and decreasing cytotoxicity in combination with 5-Fu.Resveratrol (Res) is a phytoalexin produced by the enzyme stilbene synthase in response to environmental stress such as vicissitudes in climate, exposure to ozone, sunlight and heavy metals, and infection by pathogenic microorganisms. Numerous studies show that resveratrol has the ability in protecting against many diseases including cancer. Resveratrol can potentially interfere with all three major stages of carcinogenesis (initiation, promotion and progression). The anti-tumor properties of resveratrol have been associated with its antioxidant activity and ability to affect cell growth, inflammation, apoptosis, angiogenesis, and invasion. It has the ability to affect many gene expression via many pathways, these include tumor suppressors p53 and Rb, cell cycle regulators Cyclins and many apoptotic and survival regulators. These in vitro and in vivo studies provide a rational basis in support of using resveratrol in human cancer chemoprevention, in a combinatorial approach with other chemotherapeutic drugs for the highly efficient treatment of drug refractory tumor cells.Skin cancer, especially the nonmelanoma skin cancers (NMSC), is the most common form of human cancer. It is estimated that over 1 million new cases occur annually. The annual rates of all forms of skin cancer are increasing each year, representing a growing public concern. Esophageal cancer is the sixth leading cause of cancer mortality. China is among the areas of high incidence of esophageal cancer. Given the high incidence of the two cancers, we chose human epidermal cancer cell lines A431and esophageal squamous cell cancer TE-1 to determine the individual and combined effects of resveratrol and 5-Fu on cell growth, cell cycle, and apoptosis. We also investigated apoptotic pathways that may be involved in the interactions of resveratrol and 5-Fu.PART 1 The Growth Inhibition and Cytotoxicity of 5-Fu Combined with Resveratrol on the of A431 and TE-1 Cell LinesObjective: To study the effects of 5-Fu combined with Res on the growth and necrosis of A431 and TE-1 cell linesMethods: The effects of 5-Fu combined with Res on the viability of A431 and TE-1 cell lines were evaluated by MTT assay, growth curves assay and clonogenic assay. The inhibitory effect of combination of the two drugs was analyzed by the method of Chou and Talalay, which is a quantitative measure of the degree of drug interaction between two agents. Cytotoxicity induced by 5-Fu combined with Res on A431 and TE-1 was measured by LDH releasing assay.Results: (1) MTT assay showed that 5-Fu decreased cell viability in a concentration-dependent and time-dependent manner. Res at higher concentrations (no less than 10μmol/L) also showed an inhibition on cell viability in a concentration-dependent and time-dependent manner. Combination of 5-Fu with Res was more effective than either alone at most concentrations. Decreased the median inhibitory concentration (IC50) to A431 cells from 322.89μM to 71.99μM (5-Fu) and 174.58μM to 37.56μM (Res), respectively. And decreased IC50 to TE-1 cells from 480.83μM to 151.87μM (5-Fu) and 248.64μM to 69.42μM (Res), respectively. The results indicated a synergistic anti-tumor effect of combining 5-Fu and Res. The inhibitory effect of combination of the two drugs was assessed by the MTT assay. After treated with 5-Fu with or without Res for 48 h, the inhibition rates were analyzed by the method of Chou and Talalay. The CI value was determined at effective concentration (EC) of 5-Fu plus Res that caused minimal inhibition (10%, EC10) and maximal inhibition (95%, EC95). 5-Fu and Res acted synergistically when both agents inhibited cell viability by 50% in A431 (CI=0.438) and TE-1(CI=0.595) .The CI values were <1 when the fractions affected were higher than 0.2, which indicated that combination of 5-Fu and Res had a synergistic inhibitory effect on the viability of A431 and TE-1 cells across the broad range of fraction affected (20-95% cell death). (2) The growth rate of cells was reduced by 5-Fu or Res, compared with the control group. When 5-Fu combined with Res, the growth inhibitory effect became even more pronounced and resulted in almost complete inhibition of cell growth throughout the assay period (day 1~day 9). (3) In vitro clonogenic assays indicated that cells could shape clone at day 14. The clone formation rate of A431 and TE-1 was 17.60% and 26.75% respectively in control group, which was 5.42% and 10.13% in Res-treated group and 1.07% and 2.23% in 5-Fu treated group. There is almost no clone exists when exposed to combination of 5-Fu with Res in both A431 and TE-1 cells. The differences among the treated group were statistically significant. (4) LDH activity assay demonstrated that necrosis pathway involved in the 5-Fu and Res induced A431 and TE -1 cell death after 24h and 48h, respectively. 5-Fu plus Res also showed a stronger effect in necrosis compared with 5-Fu and Res used solely in a time-dependent manner.Conclusions: (1) 5-Fu and Res time-dependently and concentration-dependently suppressed the viability of A431 and TE-1 cells. Combination of two drugs showed a statistically significant increase in inhibition of A431 and TE-1 cells. (2) 5-Fu and Res can reduce the growth rate and colony formation of A431 and TE-1cells. The difference between combination group and group of sole medication was statistically significant. (3) Necrosis pathway was involved in the 5-Fu induced cell death at that time of 48h, and it was not until 72h that necrosis pathway was involved in the Res induced cell death. When used in combination, 5-Fu and Res began to show a significant stronger effect from 24h in a time-dependent manner.Part 2 The Effects of 5-Fu Combined with Resveratrol on the Cell cycle and protein related to proliferation of A431 and TE-1Cell LinesObjective: To study the effects of 5-Fu Combined with Res on the Cell cycle and protein related to proliferation of A431 and TE-1Cell Lines.Methods: Cell-cycle distribution of A431 and TE-1 was determined by fluorescence -activated cell sorting (FACS). Proteins related to cell cycle and proliferation were identified by Western-blot.Results: (1) Treatment of A431 and TE-1with Res for 24h~72h led to the accumulation of the S-phase cell population, which was accompanied by the reduction of the ratio of G1-cells compared with the control group (P<0.05) . Compared with control group, the S-Phase fraction of A431 increased from 29.2±1.4% to 65.3±2.5% with 50μM Res (P <0.01). Res had the same effect on TE-1 cell-cycle progression with S-phase fraction being increased from 26.2±3.2% to 89.3±3.7% with 70μM Res (P <0.01). Meanwhile, compared to the control group, some of the G1-phase cells were increased slightly in 5-Fu- treated group and the combination group, but the changes were not statistically significant up to 24h treatment. The cells at G0/G1 phase increased significantly while compared with control group after treatment of A431 and TE-1 cells with 5-Fu with or without Res for 48h, but the difference between 5-Fu group and combination group was not statistically significant. (2) Analysis of the protein level of the key cell cycle regulators substantiated the results of the flow cytometry. The cell cycle arrest in S-phase induced by resveratrol coincided with decrease of the levels of CyclinD1 and PCNA.Conclusions: (1) Treatment of A431 and TE-1 cells with 5-Fu with or without Res increased the population of cells in G0/G1 phase. Treatment of A431 and TE-1 cells with Res increased the population of cells in S-phase. In the light of the mechanism of 5-Fu,we hypothesize that one reason of the synergistic interaction between resveratrol and 5-Fu is that resveratrol enhances the sensitivity of tumor cells to 5-Fu by increasing numbers of S-phase cells. (2) 5-Fu and Res inhibited the proliferation of A431 and TE-1 cells by stagnating the cells at different phase. Decreasing in expression of the CyclinD1 and PCNA confirmed that possibility.Part 3 Effects of 5-Fu with or without Resveratrol on the Apoptosis of A431 and TE-1 cell LinesObjective: To study the effects of 5-Fu with or without Res on apoptosis of A431 and TE-1cell lines and the underlying mechanism. Methods: Apoptosis of A431 and TE-1 was determined by inverted microscope, DAPI staining, gel electrophoresis of DNA fragment analysis and AnnexinV-PI assay. Intracellular Ca2+ concentration was determined and Western-blot (Proteins related to apoptosis) was used to study the underlying mechanisms of 5-Fu with or without Res on the apoptosis of A431 and TE-1 cell lines.Results: (1) The morphology of A431 and TE-1 cells was observed by invert microscope and fluorescence microscopy following DAPI staining. After treated with 5-Fu with or without Res, the loss of cell membrane asymmetry and attachment and cell shrinkage were found in A431 and TE-1 cells by invert microscope. Other morphologic changes such as nucleuspyknosis, chromatin condensation and apoptotic body formation were found in the cells incubated continuously with 5-Fu with or without resveratrol for 48 h by DAPI staining. Much more fragmented nuclei and typical morphological changes of apoptosis were observed in the cells treated with 5-Fu and resveratrol in combination than that in the cells treated with the agents alone. (2) The treatment-induced apoptosis was also apparent from the changes detected by DNA Ladder. There appeared to be differences in the amount of DNA laddering formed following different treatment. A distinct banding pattern of fragmented DNA was found easily in cells treated with combination of 5-Fu and resveratrol, while smaller amount of fragmented DNA was seen in the cells treated with resveratrol alone and DNA ladders were indistinct following 5-Fu treatment. (3) After treated with 5-Fu, the percent of apoptotic cells were assessed by Annexin V-FITC and propidium iodide staining, followed by flow cytometric analysis. Either 5-Fu or resveratrol alone caused an apparent increase in the percentage of early apoptotic cells compared to control (P<0.05). Untreated A431 cells show less than 6% spontaneous apoptosis (early apoptotic cells); however, incubation with 5-Fu (70μmol/L) or resveratrol (50μmol/L) for 24 h induced 19% and 9.3% apoptosis in A431, respectively. When cells incubated with both 5-Fu (70μmol/L) and resveratrol (50μmol/L), there was a significant increase in the percentage of cells undergoing apoptosis (approximately 34.2% apoptosis rate) (P<0.01 vs sole medication). Untreated TE-1 cells show less than 5% spontaneous apoptosis (early apoptotic cells); however, incubation with 5-Fu (150μmol/L) or resveratrol (70μmol/L) for 24 h induced 11% and 7.5% apoptosis in TE-1, respectively. When cells incubated with both 5-Fu (150μmol/L) and resveratrol (70μmol/L), there was a significant increase in the percentage of cells undergoing apoptosis (approximately 24.2% apoptosis rate) (P<0.01 vs sole medication). (4) The increase of [Ca2+] was involved in the apoptotic induction and at the upstream of the signal pathway. Both 5-Fu and Res alone increased [Ca2+], and combination of the two drugs induced a significant increase of [Ca2+] compared with either drug alone (P<0.01). (5) To determine whether the treatment-induced apoptosis was associated with expression of apoptosis-regulating proteins, the cells were treated for 48 h with 5-Fu and/or resveratrol, and then were subjected to Western blotting. Since the expression of Bcl-2, Bax, caspases-3 and PARP has been reported to play a crucial role in apoptotic response mediated by many chemopreventive agents, the changes in the expression levels of these proteins were observed by Western blot analysis in our study. The results showed that either 5-Fu or resveratrol caused a marked down-regulation of Bcl-2 protein expression, and the combination was even more effective. Expression of pro-apoptotic proteins Bax and p53 was also significantly increased by 5-Fu and/or resveratrol. In addition, the ratio of pro-apoptotic/anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. The expression of caspase-3 and PARP proteins was down-regulated concurrently with the increase of caspase-3 and cleaved PARP expression after 48h treating with 5-Fu in combination with resveratrol, and the expression of caspase-3 and cleaved PARP was synergistically increased by the combination of the two compounds. These findings suggested that the synergistic effect on apoptosis of 5-Fu and resveratrol might be due to enhancing expression of pro-apoptotic Bax and p53 proteins and reducing expression of anti-apoptotic Bcl-2 protein. Besides, active caspase-3 was generated by either 5-Fu or resveratrol and combination treatment was more effective than either alone in both A431 and TE-1 (P<0.01).Conclusions: (1) 5-Fu with or without Rescan induced apoptosis of A431 and TE-1 cells (2) 5-Fu combined with Res showed a synergistic effect for inducing apoptosis of A431 and TE-1 cells (3)The synergistic effect in apoptosis of A431 and TE-1 cells could be associated with an increasing of p53 and ratio of Bax to Bcl-2, followed by activation of caspase-3 and PARP, suggesting that mitochondrial pathways were involved in 5-Fu/resveratrol-induced apoptosis of cancer cells.Part 4 Therapeutic effects of 5-Fu and Resveratrol alone or in combination on two-stage mouse skin carcinogenesisObjective: To study the therapeutic effects of 5-Fu with or without Res on mouse skin papilloma chemically induced by DMBA/TPA.Methods: The mouse skin papilloma model was established by 7,12- dimethylbenz (a) anthracene (DMBA) and 12-O-tetradecanoylphorbol-13- acetate (TPA),which was a classical two-stage cancer model. The expression of p53, Bax and Bcl-2 in A431and TE-1 cells was identified by western-blot method. Expression of actived- caspase-3 in mouse skin was examined by immunohistochemistry.Results: (1) Treatment with 5-Fu with or without Res increased the pro-apoptotic proteins Bax and p53, and decreased the anti-apoptotic protein Bcl-2 expression. In addition, the ratio of pro-apoptotic / anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. (2) Active caspase-3 was generated by either 5-Fu or resveratrol and 5-Fu in combination with Res was more effective than either alone.Conclusions: (1) 5-Fu in combination with Res produced visible effects in curing the epidermal carcinoma of mice and reduced skin irritation of 5-Fu. (2) The synergistic effect in apoptosis of mice epidermal carcinoma may be associated with an increasing of p53 and ratio of Bax to Bcl-2.(3)5-Fu in combination with Res may induce apoptosis of mice epidermal carcinoma through activating much more activation caspase-3 than the group of sole medication.CONCLUSIONS1 Res in combination with 5-Fu inhibited synergistically growth of A431 and TE-1 cells in a concentration-dependent and time-dependent manner. Necrosis pathway was involved in the cell death induced by 5-Fu and Res in combination.2 Treatment with Res increased the population of cells in S-phase of A431 and TE-1 cells, which enhanced the sensitivity of tumor cells to 5-Fu. Treatment of 5-Fu with or without Res increased the population of cells in G0/G1 phase of A431 and TE-1 cells.3 Res combined with 5-Fu showed a synergistic effect on apoptosis of A431 and TE-1 cells. The increase of [Ca2+]i induced by 5-Fu combined with Res was involved in the apoptotic induction and at the upstream of the signal pathway. The synergistic effect on apoptosis of A431 and TE-1 cells may be associated with an increasing of p53 and ratio of Bax to Bcl-2, followed by activation of caspase-3 and PARP, suggesting that mitochondrial pathways are involved in 5-Fu/resveratrol-induced apoptosis of cancer cells.4 There was an increase of the pro-apoptotic proteins Bax and p53, and decrease of the anti-apoptotic protein Bcl-2 expression after treatment with 5-Fu with or without Res in the tumor-bearing mice model. In addition, the ratio of pro-apoptotic/ anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. Active caspase-3 in the cells of mice skin was generated by either 5-Fu or resveratrol and 5-Fu in combination resveratrol was more effective than either alone.
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