| Backgroud:Esophageal cancer(EC) is the most common cancer of upper gastrointestinal tract. The incidence of EC in China is high, which is one of the leading causes of cancer-related deaths. Prognosis of this disease is poor. It’s usually diagnosed at metastatic stage, thus many patients lose the opportunity of surgical therapy and have to accept radiation therapy or chemotherapy. However, these treatment methods generally have obvious side effects and inadequately cure the disease, therefore new treatment options are desperately needed. Pterostilbene(PTE) is a natural dimethylated analog of resveratrol derived from blueberries, grapes and tree wood. Similar to resveratrol, PTE, whose biological activity is higher, has diverse pharmacological activities, such as anti-oxidant, anti-inflammatory and anti-cancer. The anticarcinogenic effects of pterostilbene have been confirmed in many cancer types, including lung cancer, breast cancer, liver cancer, prostate cancer, and leukemia, whereas the effects of PTE on human esophageal cancer haven’t been elucidated.The endoplasmic reticulum(ER) is an essential intracellular organelle with multiple roles, including Ca2+ storage, the fold, translocation and modification of nascent proteins. Endoplasmic reticulum stress(ERS), which result from nutrient deprivation, hypoxia, Ca2+ imbalance or perturbation of protein glycosylation, is caused by disturbances in the structure and function of the ER. The disturbances will elicit the accumulation of unfolded or misfolded proteins in the ER and activation of the unfolded protein response(UPR) pathway. Under normal conditions, UPR can stimulate unfolded or misfolded proteins degradation, and restore the function of ER. However, when normal ER function can’t be restored, it wil trigger apoptosis. Importantly, studies have shown that resveratrol can induce the apoptosis of various types of cancer cells through regulating ERS signaling. However, the role of ERS signaling in the anticancer activity of PTE has not been examined.We presume that PTE has the anticancer effects on esophageal cancer, and the anticancer activity may be related to apoptosis induced by ERS. We will study the anticancer effects of pterostilbene against human esophageal cancer cells in vitro and in vivo, then further discuss the role of apoptosis induced by ERS in it. This study can not only provide a new method for chemotherapy and mechanism research of esophageal cancer, but also offer theoretical basis for illuminating ERS as a therapeutic target of cancer.Objective:1. To explore whether PTE has anticancer effects on human esophageal cancer cell.2. To explore whether PTE exerts anticancer effects on esophageal cancer cell viapoptosis induced by ERS.Methods:Part 1: Exploring the effects of PTE on human esophageal cancer cell EC109 in vitro.1. Effects of PTE treatment on the viability and cell morphology of EC109 cells. The cells were divided into 4 groups: Control and PTE 50, 100, 150μM. The four groups were respectively administered for 12, 24 and 36 h. Cell Counting Kit-8(CCK-8) was used to measure the cell viability, and inverted microscope was used to observe the morphology ofEC109 cells.2. Effects of PTE treatment on the apoptosis of EC109 cells. The cells were divided into 4 groups: Control and PTE 50, 100, 150μM. The cells were treated for 24 h. Flow cytometry was used to measure the cell apoptotic index.3. Effects of PTE treatment on the adhesion abilities of EC109 cells. The cells were divided into 4 groups: Control and PTE 5, 10, 15μM. The cells were treated for 24 h. Adhesion assay was used to measure the adhesive rate of cells.4. Effects of PTE treatment on the migration abilities of EC109 cells. The cells were divided into 4 groups: Control and PTE 5, 10, 15μM. The cells were treated for 24 h. Cell culture wound-healing assay was used to measure the migration of cells.5. Effects of PTE treatment on the ERS- and apoptosis-related proteins of EC109 cells. The cells were divided into 4 groups: Control and PTE 50, 100, 150μM. The cells were treated for 24 h. Western-bolt was used to detect the expression of ERS-related proteins(GRP78, ATF6, p-PERK, p-e IF2α, CHOP) and apoptosis-related proteins(Bcl2, PUMA).Part 2: Exploring the effects of PTE on human esophageal cancer cell EC109 in vivo.1. Effects of PTE treatment on the sizes of EC109 cell tumor xenografts. The mice were divided into 3 groups: Control, PTE 100mg/kg, PTE 200mg/kg. After EC109 cell tumor xenografts established, PTE was administered intraperitoneally(5 days/week). The tumor sizes were measured every 3 days, and on 20 th day, the tumors were excised and the tumor sizes were measured.2. Effects of PTE treatment on the ERS- and apoptosis-related proteins of EC109 cell tumor xenografts. After the tumors were excised from athymic nude mice, western-bolt was used to detect the expression of ERS-related proteins(GRP78, CHOP) and apoptosis-related proteins(Bcl2, PUMA).Part 3: Exploring the effects of ERS on the anticancer effects of pterostilbene against human esophageal cancer cell EC109 in vitro.1. Effects of PTE combined with CHOP si RNA on cell viability and morphology of EC109 cells. The cells were divided into 4 groups: ①Control si RNA; ②CHOP si RNA;③Control siRNA+PTE 100μM; ④CHOP si RNA+PTE 100μM. The EC109 cells were first treated with si RNA transfection and then subjected to PTE(100μM) treatment for 24 h. CCK-8 was used to measure cell viability, and inverted microscope was used to observe the morphology of EC109 cells.2. Effects of PTE combined with CHOP siRNA on the ERS- and apoptosis-related proteins of EC109 cells. The EC109 cells were first treated according to step 1, and then western-bolt was used to detect the expression of ERS-related proteins(CHOP) and apoptosis-related proteins(Bcl2, PUMA).3. Effects of PTE combined with thapsigargin(THA) on cell viability and morphology of EC109 cells. The cells were divided into 4 groups: ①Control; ②THA 1μM; ③PTE 100μM; ④THA 1μM+PTE 100μM. The cells were treated for 24 h. CCK-8 was used to measure the cell viability, and inverted microscope was used to observe the morphology of EC109 cells.4. Effects of PTE combined with THA on the ERS- and apoptosis-related proteins of EC109 cells. The EC109 cells were first treated according to step 3, and then western-bolt was used to detect the expression of ERS-related proteins(CHOP) and apoptosis-related proteins(Bcl2, PUMA).Results:Part 1: Exploring the effects of PTE on human esophageal cancer cell EC109 in vitro.After treated with PTE(0, 50, 100, 150μM) for 12, 24 and 36 h, the EC109 cells shrank, attached cells decreased, and the cell viability was inhibited in a dose- and time-dependent manner(P<0.01). After the treatment with PTE(0, 5, 10, 15μM) for 24 h, the adhesive and migratory abilities of EC109 cells declined(P<0.01). After the treatment with PTE(0, 50, 100, 150μM) for 24 h, the apoptotic index of EC109 cells were increased(P<0.01), the expressions of GRP78, ATF6, p-PERK, p-e IF2α and CHOP were upregulated(P<0.01), the anti-apoptosis proteins Bcl2 decreased(P<0.01) and the pro-apoptosis proteins PUMA increased(P<0.01).Part 2: Exploring the effects of PTE on human esophageal cancer cell EC109 in vivo.After EC109 cell tumor xenografts established, PTE(0, 100, 200mg/kg) was administered intraperitoneally for 20 days, the tumor sizes decreased obviously in dose-dependent manner(P<0.01), the ERS-related proteins(GRP78, CHOP) were upregulated(P<0.01), the anti-apoptosis proteins Bcl2 decreased(P<0.01) and the pro-apoptosis proteins PUMA increased(P<0.01).Part 3: Exploring the effects of ERS on the anticancer effects of pterostilbene against human esophageal cancer cell EC109 in vitro.1. The EC109 cells were first treated with siRNA transfection and then subjected to PTE(100 μM) treatment for 24 h. CHOP si RNA transfection significantly decreased CHOP expression in EC109 cells(P<0.01, compared with the control si RNA group). The combination of CHOP si RNA+PTE significantly increased cell viability(P<0.01, compared with the control si RNA+PTE group). CHOP si RNA alone had no effect on the cell viability compared with the control si RNA group(P>0.05). Additionally, PUMA was downregulated, while Bcl2 was upregulated, in the CHOP si RNA+PTE group(P<0.01, compared with the control si RNA+PTE group).2. The EC109 cells were incubated with PTE and THA for 24 h. The cytotoxic effects of PTE on EC109 cells were strengthened(combination index<1). ERS-related proteins(CHOP) were upregulated(P<0.01), the anti-apoptosis proteins Bcl2 decreased(P<0.01) and the pro-apoptosis proteins PUMA increased(P<0.01).Conclusions:PTE has anticancer effects on human esophageal cancer cell EC109 in vitro and in vivo, which can inhibit the cell viability, decrease the adhesive and migratory abilities and induce the cell apoptosis. And the anticancer effects of PTE are dependent on apoptosis induced by ERS. |
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