| NHL is the most common hematological neoplasm in the United States, and accounts for4%of all malignant tumors diagnosed each year. DLBCL is the most prevalent subtype of NHL worldwide and represents up to40%of all NHL cases among adults in the western world, and frequent in developing countries. DLBCL is the heterogeneous disease with variable cytogenetics and immunophenotype, as well as clinical features. Although the initial standard therapy with rituximab in combination with CHOP (R-CHOP) chemotherapy has improved outcomes for patients with DLBCL, there are still some patients who are refractory to initial treatment or relapse after standard therapy. Therefore, the discovery of novel and alternative therapeutic approaches is urgent.In ancient China, traditional Chinese medicine (TCM) has been used to treat malignant tumor and has been proven to be effective for a long time. And arsenic included As2S2, As4S4and As2O3. In1996, As2O3was been firstly used to induce APL differentiation and apoptosis by the institutes of Shanghai hematonosis. Consequently, arsenic drugs have become a’hot topic’and have attracted increased attention in regards to malignant hematological neoplasm.Realgar has been found to induce both apoptosis and differentiation simultaneously in ATRA-sensitive NB4and ATRA-resistant MR2PML-RARa+APL cell lines. Previous studies demonstrated that realgar induced apoptosis of HL-60, NB4and K562cell lines, which was associated with CD95/CD95L and MAPK pathway, inhibition of telomerase activity and decreased expression of BCL-2and PNAS-2. In addition, realgar induced the differentiation of HL-60cell line via not only the enhancement of the activity of serine/threonine protein phosphatase typel (PP1) and type2A (PP2A) but also oxidative stress and stress-related mitochondrial transmembrane potential (MTP).In addition, previous studies have demonstrated that arsenic induced apoptosis among in NHL cells by means of different mechanisms. They revealed that As2O3inhibited proliferation and induced apoptosis in the human Burkitt lymphoma cell line Raji and in the human T lymphoma cell line Jurkat through cell cycle arrest, decrease in respiratory function and MTP, downregulating the expression of MCL-1and subsequently activating caspase-3. However Jurkat cells were less sensitive to As2O3-induced apoptosis than Raji cells, as Jurkat cells express high levels of glutathione S-transferase P1-1(GSTP1-1). At the same time, realgar induced apoptosis of human T lymphocyte leukemia cell line CEM apoptosis through cell cycle arrest in the G2/M phase, a decrease in the expression of Bcl-2and an increase in APO2.7protein expression. A recent study demonstrated that As2O3inhibited the growth of mantle cell lymphoma (MCL) and inducesd apoptosis through a decrease in cyclinD1expression and increase in the expressin of apoptosis-related molecules.To enhance therapeutic efficacy and reduce adverse effects, it is common to combine multiple drugs to deal with diseases. Realgar-Indigo naturalis formula (RIF) was been constituted with realgar as a sovereign drug, whereas Indigo naturalis and Salvia miltiorrhiza as minister drugs. And further research demonstrated that the components of RIF were realgar and indigo with arsenic sulfide and indirubin as major ingredients. Realgar was known to be able to induce apoptosis in human T cell lymphoma cell (CEM) and human B cell lymphoma (Raji). Furthermore, indirubin was proven to induce apoptosis in human T cell lymphoma cell (Jurkat, CEM) and human B cell lymphoma (IM9, Reh6).But there was no study on whether indirubin and As2S2alone or in combination had effects in DLBCL cells. For the first time, our present manuscript focuses on indirubin and As2S2alone or in combination on the proliferation and apoptosis of DLBCL cell lines and its mechanism in an attempt to seek a more effective combination therapy scheme for DLBCL. Part Ⅰ Arsenic Disulfide Induces Apoptosis of Human Diffuse Large B-Cell Lymphoma Cells and The MechanismObjective:DLBCL is the most prevalent subtype of NHL worldwide and represents up to40%of all NHL cases among adults in the western world, and frequent in developing countries. DLBCL is the heterogeneous disease with variable cytogenetics and immunophenotype, as well as clinical features. Although the initial standard therapy with rituximab in combination with CHOP (R-CHOP) chemotherapy has improved outcomes for patients with DLBCL, there are still some patients who are refractory to initial treatment or relapse after standard therapy. Therefore, the discovery of novel and alternative therapeutic approaches is urgent. Currently, arsenic drugs have become a’hot topic’and have attracted increased attention in regards to malignant hematological neoplasm and other solid tumors. To investigate the effect of As2S2on the proliferation and apoptosis of DLBCL cell lines LY1and LY8in an attempt to find potential mechanism.Materials and Methods:1. Co culture with DLBCL cells and As2S22. CCK-8method to detect cell proliferation3. Flow cytometry to evaluate cell apoptosis4. RNA extraction and RT-PCR5. Protein extraction and western blot analysis6. Statistical analysisResults:1. We found that the DLBCL cells viability significantly decreased by As2S2at24h,48h and72h. Along with increasing As2S2concentration, the DLBCL cells viability was notably reduced compared with the control group, and was statistically significant (P<0.05).2. The apoptotic rates of DLBCL cells were significantly increased at24h,48h and72h with increasing As2S2concentration, and were statistically significant (P<0.05). 3. The quantitative real-time PCR results showed that the expression levels of Bax/Bcl-2ratio and caspase-3mRNA were up-regulated in As2S2-treated DLBCL cells.4. Western blot revealed that As2S2could down-regulate the expression of procaspase-3and up-regulate the ratio of Bax/Bcl-2. Our study also showed that21-KDa Bax was proteolytically cleaved into the more apoptotic18-KDa Bax in DLBCL cells exposed to As2S2at a concentration of10μM.Conclusions:1. As2S2could inhibit proliferation and induce apoptosis of LY1and LY8cells in a concentration-and time-dependent manner.2. The effect was partly due to the induction of mitochondria-dependent apoptosis involving Bax cleavage.Part Ⅱ Enhancing Effects of Indirubin on Arsenic Disulfide-Induced Apoptosis of Human Diffuse Large B-Cell Lymphoma Cells and The MechanismObjective:To enhance therapeutic efficacy and reduce adverse effects, it is common to combine multiple drugs to deal with diseases. RIF was been constituted with realgar as a sovereign drug, whereas Indigo naturalis as a minister drug. Indirubin and its derivatives have been made great progress on the treatment of chronic myeloid leukemia (CML). Extensive research has been performed to study the mechanism of their anti-tumor activity. To investigate the enhancing effect of indirubin on arsenic disulfide (As2S2) on the proliferation and apoptosis of DLBCL cells LY1and LY8in an attempt to find a better combination therapy scheme.Materials and Methods:1. DLBCL cells cultured with indirubin and As2S2alone or in combination2. CCK-8method to detect cell proliferation3. Flow cytometry to evaluate cell apoptosis4. RNA extraction and RT-PCR5. Protein extraction and western blot analysis 6. Statistical analysisResults:1. We found that the DLBCL cells viability had no significant change at24h,48h and72h with increasing indirubin concentration.2. Meanwhile the apoptotic rates of DLBCL cells weren’t significantly increased at48h with increasing indirubin concentration.3. Along with the combination of indirubin and As2S2, the viability and apoptotic rate of DLBCL cells were both notably changed compared with the As2S2-treated group, and was statistically significant (P<0.05).4. The quantitative real-time PCR results showed that indirubin had no enhancing effect on the expression levels of Bax/Bcl-2ratio and caspase-3mRNA.5. Western blot revealed that indirubin had enhancing effect on the expression levels of Bax/Bcl-2ratio and caspase-3protein. Our study also showed that21-KDa Bax was proteolytically cleaved into the more apoptotic18-KDa Bax in DLBCL cells exposed to the combination of indirubin and As2S2.Conclusions:1. Indirubin alone couldn’t inhibit proliferation and induce apoptosis of LY1and LY8cells.2. The combination of indirubin and As2S2yielded enhancing effects. Therefore our data demonstrated that As2S2served as the principal component, whereas indirubin served as the adjuvant ingredient on the anti-tumor activity.3. The enhancing effect was partly due to the induction of mitochondria-dependent apoptosis involving Bax cleavage. |
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