gossypol-acetic-acid and Multiple-Myeloma

Multiple myeloma, the second most common haematological malignancy in the U.S., is currently incurable. Disruption of the intrinsic apoptotic pathway by BCL2 and MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Thus, we investigated the anti-tumour effects of a pan-BCL2 inhibitor, AT-101, which has high binding specificity for BCL2 and MCL1 in preclinical models of plasma cell cancers (Multiple myeloma and Waldenström macroglobulinaemia). Gene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of BCL2 family members. AT-101 was able to downregulate BCL2 and MCL1 in all plasma cell cancer models and induced apoptotic cell death in a caspase-dependent manner by altering mitochondrial membrane permeability. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). NanoString nCounter mRNA quantification and Ingenuity Pathways Analysis revealed differential changes in the CCNA2, FRZB, FYN, IRF1, PTPN11 genes in LDA-treated cells. In summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Dexamethasone; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Profiling; Gossypol; Humans; Lenalidomide; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Thalidomide; Waldenstrom Macroglobulinemia

Gossypol is a naturally occurring polyphenolic compound extracted from cotton plants. Recent studies revealed that gossypol is a non-peptidic small molecule inhibitor of Bcl-2/Bcl-xl. The aim of the present study was to investigate the induction of apoptosis and antitumor effects of gossypol acetate in multiple myeloma and the possible mechanism(s) of action. Our results showed that gossypol acetate resulted in a dose- and time-dependent inhibition of multiple myeloma cell proliferation, with an IC50 value to both U266 and Wus1 cells at 2.4, 2.2 µM at 48 h after treatment. Gossypol acetate effectively induced the apoptosis of multiple myeloma cells as demonstrated by typical morphological changes, DNA ladder formation and increase in the percentage of cells in subdiploid peak. Furthermore, colorimetric assays showed activation of both caspase-3 and caspase-9. Bcl-2 and Bcl-xl expression was decreased by 86.5±1.2% and 35.9±3.6%, respectively, after treatment with gossypol acetate at 25 µmol/l for 24 h. Preliminary studies in vivo showed that a growth inhibition (T/C) of 30.9% (gossypol acetate 40 mg/kg) was obtained in Balb/C mice bearing Wus1 cells. In addition, there was no body weight loss for the treated group in comparison with the vehicle mice. Our results demonstrated that the potent inhibitor of Bcl-2 and Bcl-xl gossypol acetate had significant antiproliferative and antiapoptotic effects on multiple myeloma cells in vitro and in vivo. Gossypol acetate may represent a promising new anticancer agent with a novel molecular mechanism and warrants further investigation as a single agent, or in combination with other chemotherapeutics, for human multiple myeloma with Bcl-2 overexpression.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gossypol; Humans; Mice; Mice, Inbred BALB C; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Small Molecule Libraries

This study was aimed to investigate the apoptosis effect of gossypol acetic acid on classic human multiple myeloma RPMI8226 cell line in vitro and its mechanism. The inhibitory effect on proliferation of RPMI8226 cells was evaluated by means of MTT assay. Cytotoxic effect and apoptosis was identified and analyzed with the aid of transmission electron microscopy, mitochondria membrane potential (MMP) and DNA gel electrophoresis. Meanwhile, Western-blot assay was used to detect the changes of several key cell apoptosis regulatory proteins such as BAX, caspase-3 and caspase-8 in these cells before and after treatment. The results showed that low concentrations of gossypol acetic acid (> 16 micromol/L) could suppress the proliferation and induce the apoptosis in RPMI8226 cells effectively. At the same time, gossypol acetic acid could also down-regulate the mitochondrial membrane potential, up-regulate the expression of the apoptosis-related protein such as BAX and caspase-3. It is concluded that the gossypol acetic acid can selectively induce proliferation inhibition and apoptosis of multiple myeloma RPMI8226 cells with a smaller dose.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Proliferation; Gossypol; Humans; Membrane Potential, Mitochondrial; Multiple Myeloma

Bcl-2 family proteins play a critical role in malignancies by regulating the balance between cell survival and apoptosis. R-(-)-gossypol (AT-101) is a small molecule that mimics the BH3 domain of cellular Bcl-2 inhibitors and interferes with the function of prosurvival Bcl-2 proteins. We examined the cytotoxicity of AT-101 in the context of multiple myeloma, a fatal hematological malignancy.. Multiple myeloma cell lines and primary cells obtained from multiple myeloma patients were used to investigate the effects of AT-101. Cell viability, apoptosis, and apoptosis pathways were examined using conventional viability assays, flow cytometry, and immunoblots.. AT-101 was not only cytotoxic to conventional multiple myeloma cell lines, but was also effective against drug-resistant cell lines and primary multiple myeloma patient cells. Furthermore, AT-101 decreased proliferation of multiple myeloma cell lines in the presence of marrow stromal cells, indicating that this drug may overcome the protective effect of the microenvironment that is important for multiple myeloma cell proliferation and survival. Apoptosis was activated via the mitochondrial pathway in multiple myeloma cell lines treated with AT-101 as demonstrated by an increased Bax to Bcl-2 ratio, mitochondrial membrane depolarization, and caspase activation. Finally, our studies demonstrated that AT-101 exhibits potent synergy with dexamethasone, a valuable therapeutic for multiple myeloma.. These data suggest that the activity of AT-101 may be highly relevant to multiple myeloma disease biology and may represent an option for treatment of patients with this disease.

Topics: Analgesics; Apoptosis; Caspases; Cell Proliferation; Cell Survival; Dexamethasone; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Flow Cytometry; Gossypol; Humans; Immunoblotting; Mitochondria; Mitochondrial Membranes; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line; DNA; Gossypol; HeLa Cells; Humans; Isoenzymes; L-Lactate Dehydrogenase; Liver Neoplasms; Mice; Multiple Myeloma; Rats