| Background and ObjectiveMalignant pleural mesothelioma (MPM) is an asbestos-related and highly aggressive malignancy, the incidence of which is still expected to increase over the next few decades because of its long latency period (20-40years), although world-wide use of asbestos has decrease in recent years. The overall survival (OS) is poor, with median OS at approximately12months. The studies of prognosis of MPM were mainly conducted abroad. The lack of Chinese data encourages us to conduct this study to investigate whether the prognostic nutritional index (PNI), an indicator of nutrition status, affects OS in patients with MPM in the first part.The current therapies (surgery, chemotherapy, radiotherapy and trimodality therapy) don’t have ideal curative effects but have many undesirable side effects. MPM remains difficult to treat and standard therapies are still in flux. Therefore, a novel and more effective treatment strategy is urgently required.Berberine is an isoquinoline alkaloid exacted from a number of medicinal plant species such as Berberis aquifolium and Berberis aristata, with extensive pharmacological effects. Recent studies have shown that berberine exerts antitumor effects in a variety of human cancer cells such as hepatic, gastric, esophageal, breast and bladder cancer cells by suppressing proliferation, inducing apoptosis, arresting cell cycle and inhibiting cancer invasion and metastasis. Recently, some studies reported that berberine exerted antitumor effects through autophagy.However, to our knowledge, the antitumor effect of berberine has not been investigated in MPM cells. Therefore, in the second part we aimed to investigate the antitumor effect of berberine in human MPM NCI-H2452cells, and further illustrate the roles of berberine-induced apoptosis and autophagy and underlying molecular mechanisms involved in antitumor activities.First part:study of relationship between prognostic nutritional index and prognosis of MPMPurposeWe aimed to investigate whether PNI, an indicator of nutritional status, affects OS in patients with MPM.MethodsWe enrolled121patients with histologically confirmed MPM, who had successfully undergone biopsy by medical thoracoscopy in this study from October2001to January2013. Demographic, clinical and laboratory data were collected retrospectively. The PNI was calculated as10x serum albumin value (g/dl)+0.005×total lymphocyte count (per mm3) in peripheral blood. Univariate and multivariate analyses were used to identify prognostic factors.Results1. Mean pretreatment PNI was44.6±6.3. PNI was significantly associated with age, smoking habits and weight loss (P<0.05).2. Mean PNI (44.6) in our study cohort was used to divide patients into the PNI-low group (60patients [49.6%] with PNI<44.6) and the PNI-high group (61patients [50.4%] with PNI≥44.6). As of the last follow-up,77patients (63.6%) had died—31patients in the PNI-high group and46patients in the PNI-low group. Median OS of all patients was15months (range:0.3-86months). The Kaplan-Meier method and log-rank test indicated that the lower PNI value was associated with shorter OS (P=0.003). Median OS of patients in the PNI-high and PNI-low group was18and11months, respectively;1-year OS rates were72.3and45.5%in PNI-high group and PNI-low group, respectively; and the2-year OS rate was38.7%for PNI-high group and18.4%for PNI-low group.3. Survival analysis showed PNI to be an independent prognostic factor in MPM. Patients with lower PNIs (PNI<44.6) had greater risk of death than those with higher PNIs (PNI≥44.6; hazard ratio:2.290;95%confidence interval:1.415-3.706; P=0.001).ConclusionsPretreatment PNI is a novel independent prognostic factor in MPM. Patients with lower PNIs had greater risk of death than those with higher PNIs.Second part:The study on apoptosis and autophagy induced by berberine in human pleural mesothelioma cell and its underlying mechanismPurposeIn this part we aimed to investigate the antitumor effect of berberine in human MPM NCI-H2452cells, and further illustrate the roles of berberine-induced apoptosis and autophagy and underlying molecular mechanisms involved in antitumor activities.Methods1. The function and molecular mechanisms of berberine-induced apoptosis in human MPM NCI-H2452cells3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate viability of NCI-H2452cells. Hoechst33258staining and Annexin-V-PE/7-AAD double staining assay were used to evaluate apoptosis. Western blotting analysis was performed to detect the signal transduction in apoptosis activated by berberine.2. The study of berberine-induced autophagy in human MPM NCI-H2452cellsThe intracellular distribution of microtubule-associated protein1light chain3(LC3), an autophagy marker, was detected by immunofluorescence. Western blot analysis was performed to detect the expression level of autophagy-related proteins and the effects of autophagy inhibitor or inducer on berberine-induced autophagy.3. The role of autophagy in berberine-induced MPM NCI-H2452cell deathMTT assay was used to detect the effects of autophagy inhibitor3-methyladenine (3-MA) on the inhibition of cell viability induced by berberine. Cell death in response to berberine was assessed by trypan blue exclusion assay. To further explore the roles of apoptosis and autophagy in berberine-induced MPM cell death, the autophagy inhibitor3-MA, autophagy inducer rapamycin, or apoptosis inhibitor z-VAD-fmk was used to manipulate the autophagy and apoptosis induced by berberine. Annexin V-PE/7-AAD double staining assay and western blot assay were used to illustrate the interconnection between berberine-induced autophagy and apoptosis.Results:1. The function and molecular mechanisms of berberine-induced apoptosis in human MPM NCI-H2452cells(1) Berberine significantly inhibited the proliferation of NCI-H2452cells in a dose-and time-dependent manner.(2) Hoechst33258staning and Annexin-V-PE/7-AAD double staining assay showed that berberine induced apoptosis of NCI-H2452cells. After treatment with berberine at the concentration of100μM for48hours, the apoptosis rate of NCI-H2452cells increased to34.23±1.16%, while the apoptosis rate was4.55±0.96%in untreated cells (P<0.001)(3) Western blot analysis showed that berberine could induce apoptosis in NCI-H2452cells possibly through a caspase-9dependent intrinsic mitochondrial pathway.2. The study of berberine-induced autophagy in human MPM NCI-H2452cells(1) The distribution of LC3fluorescence changed from a diffuse cytosolic manner in untreated cells to a punctate manner upon berberine treatment.(2) Western blot analysis showed that an increased conversion of the normal LC3I to the autophagic form LC3II isoform was observed in NCI-H2452cells treated with berberine in a dose-and time-dependent manner. The increased conversion was attenuated by pretreatment with autophagagic inhibitor3-MA, and enhanced by autophagic inducer rapamycin, respectively. The expression of p62decreased after berberine treatment in a dose-and time-dependent manner.3. The role of autophagy in berberine-induced MPM NCI-H2452cell death(1) Berberine induced NCI-H2452cell death in a dose-dependent manner. After treatment with berberine at the concentration of200μM for48hours, the cell death rate of NCI-H2452cells increased to34.66±5.32%(P<0.001)(2) Pretreatment with pan-caspase inhibitor z-VAD-fmk attenuated the berberine-induced cell death, which suggested that apoptosis is involved in the berberine-induced cell death.(3) The berberine-induced cell death in NCI-H2452cells was enhanced by the3-MA, while it was attenuated by autophagy inducer rapamycin.(4) The inhibition of autophagy by3-MA enhanced berberine-induced apoptosis which was confirmed by Annexin-V-PE/7-AAD double staining assay.Conclusions:(1) Berberine is a potent antitumor agent for treating MPM.(2) Berberine inhibited the proliferation of NCI-H2452cells in a dose-and time-dependent manner and could induce apoptosis possibly through a caspase-9dependent intrinsic mitochondrial pathway.(3) Berberine induced autophagy in NCI-H2452cells and down-regulated the expression of p62.(4) Apoptosis is the major form of berberine-induced NCI-H2452cell death. Berberine-induced autophagy may be an adaptive response to antitumor agents and functions as a protective role in MPM cells. Inhibition of autophagy enhanced berberine-induced apoptosis. Therefore, inhibition of autophagy may be an effective treatment strategy in controlling MPM.Originality: (1) We investigated, for the first time, that PNI is a poor predictor of OS in MPM.(2) We demonstrated, for the first time, the effect of berberine on the proliferation, apoptosis and autophagy in MPM cells.(3) We investigated, for the first time, the role of autophagy in berberine-induced cell death in MPM cells, which could lay a foundation for the antitumor treatment targeted autophagy for MPM. |
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