| Esophageal squamous cell carcinoma(ESCC) is one of the most common cancer in China and the world, and the fourth most frequently cause of death in our country. According to reports, the overall five-year survival rate of ESCC patients is less than 20% after surgery. Currently, chemotherapy has become one main method of conventional cancer therapy. However, long-term use of chemotherapy drugs, such as 5-Fluorouracil(5-FU), can cause drug resistance in tumor cells and normal cells, and tissues will also suffer a considerable degree of damage. Then, cancer patients have to give up chemotherapy because of the side effects. Therefore, it is distinctly important how to protect the normal tissue from the side effects by chemotherapy and increase the sensitivity of tumor cells to chemotherapy. Recently, some studies have shown that the use of natural compounds in combination with chemotherapy drugs can not only reduce chemotherapy side effects on normal cells, but also increase the rate of tumor cell apoptosis. Therefore, it has become a focus of anti-cancer research to find one natural antitumor compound which has a wide range of targets, low toxicity and high efficiency. β-carotene, a polyene compound, which is widely found in green plants, fruits and vegetables, has been studied extensively. β-carotene, as a precursor of vitamin A in the human body, can be transformed into two molecules of vitamin A, which play an important role in cell growth, proliferation and differentiation of epithelial cell growth. Epidemiological studies confirm that regular consumption of β-carotene-rich fruits and vegetables can effectively reduce the incidence of various cancers. Studies have shown that β-carotene has antioxidant, scavenging reactive oxygen free radicals and other functions, and its anti-tumor effect may be related to the regulation of intracellular redox status. Recent studies show β-carotene can inhibit growth and induce apoptosis of various cancer cell through a variety of mechanisms, including breast cancer, colon cancer, melanoma and prostate cancer. However, few studies have directly investigated the possible influence of β-carotene on ESCC cell growth. Furthermore, it’s unclear whether β-carotene can synergistic enhance the antitumor effect of 5-FU.In the present study we aim to explore the antitumor effects of β-carotene combined with 5-FU as well as its underlying biological mechanisms in vitro and in vivo, in hoping of assessing a relatively effective and safe regimen potentiated for ESCC treatment. This topic is divided into two parts:This topic is divided into two parts:Part I: To determine the optimal dose and treatment time of β-carotene in antitumor effects and to explore the effect of β-carotene on esophagus squamous cell proliferation, apoptosis, cycle, migration and its molecular mechanism.Part II: To investigate the antitumor effect of β-carotene in combination with 5-FU as well as its underlying biological mechanisms on Eca109 cell and Eca109 xenograft mouse model in vitro and in vivo.Method: 1. To evaluate the effects of drugs on esophageal squamous cell proliferation, apoptosis, cycle, migration in vitro and to explore its underlying biological mechanisms Cell Counting Kit-8(CCK-8) assay was used to measure the effect of 5-FU and β-carotene on the cell activity. Flow cytometry(FCM) was employed to conduct cell cycle analyses. The Annexin V-fluorescein isothiocyanate /propidium iodide(FITC/PI) assay was used to detect apoptosis using FCM. Transwell assay was used to estimate the number of migrated cells. Quantitative RT PCR were used to detect the m RNA levels of the caveolin-1(Cav-1) expression. Western Blot was used to detect the expression of E-cadherin, cyclin D1 and the activity of Cav-1/AKT/NF-κB signaling pathway. 2. The antitumor effect of β-carotene in combination with 5-FU as well as itsunderlying biological mechanisms on Eca109 xenograft mouse model in vivo Tumors were induced by subcutaneous injection of Eca109 cells(5 × 106 per mice) into the lower left flank of BALB/c mice. After the tumor reached 100~200 mm3 in size, mice were randomly assigned to four groups(n=7).(a) Control, the mice were treated by an intra-peritoneal injection of 0.9% sodium chloride for two weeks(control).(b) 5 mg/kg β-carotene, mice were treated by an intragastric administration of β-carotene(5 mg/kg) dissolved in corn oil every two days for three weeks.(c) 5 mg/kg 5-FU, the mice were treated by an intra-peritoneal injection of 5-FU(5 mg/kg) every two days for three weeks.(d) β-carotene and 5-FU according to the same dose schedule as for individual treatments. Mice body weight and tumor volume measurements were performed every three days. Three weeks after treatment, tumors were excised, and weighted. The expression level of Bcl-2, PCNA, Bax and Caspase-3 and DNA damage after different treatment was detected using immunohistochemistry and Tunnel assay. HE staining was used to analyze the pathological changes in the major organs of nude mice.Results Part I The effect of β-carotene on ESCC cells and its molecular mechanism 1. The effect of β-carotene on esophageal squamous cell proliferation, apoptosis, migration and cell cycle β-carotene significantly inhibited the proliferation of esophageal cancer cells in a dose- and time-dependent manner. Cell growth decreased by approximately 50% when TE1 cells were treated with 30 μM of β-carotene for 48 h. Cell growth decreased by approximately 50% when EC1 and Eca109 cells were treated with 50 μM of β-carotene for 48 h. Conversely, β-carotene could not exert inhibitory effect on the growth of Het-1A cells even at high concentration(50 μM). Compared with the control group, β-carotene can significantly promote cell apoptosis of esophageal squamous carcinoma cell lines TE1, EC1 and Eca109(P<0.05), arrested cell cycle in G0/G1 phase(P<0.05), and reduced cell migration(P<0.05). In addition, the level of E-cadherin expression was significantly increased compare to the control group, and the level of Cyclin D1 expression was significantly decreased(P<0.05). 2. The mechanism of inhibition effect of β-carotene on ESCC cell proliferationQuantitative RT-PCR assay showed that the m RNA levels of the Cav-1 was significantly decreased. Western blot assay showed that the level of Cav-1 、p-AKT(Ser473)、p-NF-κBp65(Ser536) and Bcl-2 expression remarkably decreased after β-carotene treatment for 48 h, the expression level of pro-apoptotic factor, Caspase-3 and Bax, was remarkably upregulated. Part II The effect of β-carotene in combination with 5-FU on Eca109 cells and its underlying biological mechanisms in vitro and in vivo 1. The effect of β-carotene combined with 5-FU on cell proliferation, apoptosis, migration and cell cycle in Eca109 cells CCK-8 assay showed that the inhibitory effect of 5-FU(30 μM) with β-carotene(30 μM) on Eca109 cell was potent, the inhibitory ratio of 5-FU(30 μM) with β-carotene(30 μM) on Eca109 cell growth was 60%±3.17%, while the inhibitory ratio of 5-FU(30 μM) or β- carotene(30 μM) alone on Eca109 cell growth was 40%±2.03%、22%±3.01%, respectively. Eca109 cells treated with 5-FU(30 μM) or/and β-carotene(30 μM) had a typical morphological characteristic of apoptosis, such as volume shrinkage, separated from the surrounding cells, irregular. Moreover, the apoptosis rate of drug combination group was significantly higher than single agent group and the control group. In addition, the drug combination group can be more effective in reducing cell migration and enhancing the expression of E-cadherin in comparison to single agent group. 2. The mechanism of inhibition effect of β-carotene combined with 5-FU on the Eca109 cell proliferation Western blot assay showed that β-carotene and/or 5-FU can inhibit the activity of Cav-1/AKT/NF-κB signaling pathway. The expression of p-AKT, p-NF-κBp65(Ser536) and Bcl-2 were down-regulated, and the expression of Bax, caspase-3 were up-regulated after β-carotene and/or 5-FU treatment. Moreover, the effect of drug combination group was significantly higher than single agent group. 3. β-carotene synergistically enhances the anti-tumor effect of 5-FU on ESCC in vivo Three weeks after treatment, the tumor volume was measured using Vernier calipers. Then tumors were excised, and weighted. After treatment tumor size andweight were: control group,(1198±156) mm3(0.38±0.08)g; β-carotene group,(949±92) mm3,(0.28±0.01) g; 5-FU group,(652±88) mm3,(0.20±0.05) g; β-carotene combined with 5-FU group,(408±61) mm3,(0.12±0.03) g,respectively. Compared with the control group, β-carotene or 5-FU significantly inhibited tumor growth. Furthermore, β-carotene plus 5-FU treatment was more effective than 5-FU treated group(P <0.05). Immunohistochemistry showed that 5-FU or β-carotene treatment significantly down-regulated the expression of Bcl-2, PCNA, and up-regulated the expression of Bax, Caspase-3, compare with control group. In combined treatment groups, the level of Bax and Caspase-3 expression were significantly increased, compare with 5-FU or β-carotene alone groups. In comparison to control group(5.02±0.81%), the percentage of the TUNEL positive apoptotic cells were increased in 5-FU(23.03±2.67%) or β-carotene(12.15±1.44%)treatment group, and showed a further decrease in the combined treatment groups(38.61±2.45%)(P<0.01). HE staining results indicated that 5-FU or/and β-carotene was effective in inhibiting the growth of transplanted esophagus cancer tumors with low toxicity in vivo.Conclusion: 1. β-carotene could inhibit ESCC cell proliferation, induce cell apoptosis and arrest cell cycle in G0/G1 phase by inhibiting the activity of the Cav-1/AKT/NF-κB signaling pathway. 2. β-carotene synergistically enhanced the anti-tumor effect of 5-FU on Eca109 cells via Cav-1/AKT/NF-κB pathway. 3. β-carotene combination with 5-FU significantly suppressed the growth of Eca109 esophageal cancer xenograft tumors and induced cell apotosis. |
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