Apogossypolone (ApoG2)

Apogossypolone is an inhibitor of Bcl-2, Mcl-1 and Bcl-XL with Ki values of 35nM, 25nM and 660nM, respectively [1].

The MTT-based cell cytotoxicity assay shows that apogossypolone has an anticancer activity with IC50 values of 1, 2 and 3μM, respectively in PC-3, DU-145 (human prostate cancer cell lines) and MDA-MB-231(human breast cancer cell line). Apogossypolone is also found to inhibit the colony formation of DU-145 cells. The mechanism is that apogossypolone binds to Bcl-2 and prevents its association with BH3-only pro-apoptotic proteins, leading the pro-apoptotic proteins to participate in the apoptotic response [2].

Besides prostate cancer and breast cancer, apogossypolone is also potent in follicular lymphoma. Apogossypolone significantly inhibits the cell growth via inducing apoptosis in WSU-FSCCL cell line with IC50 value of 109.2nM at 72h [3].

References:
[1] Zhang XQ, Huang XF, Hu XB, Zhan YH, An QX, Yang SM, Xia AJ, Yi J, Chen R, Mu SJ, Wu DC. Apogossypolone, a novel inhibitor of antiapoptotic Bcl-2 family proteins, induces autophagy of PC-3 and LNCaP prostate cancer cells in vitro. Asian J Androl. 2010 Sep;12(5):697-708.
[2] Zhan Y, Jia G, Wu D, Xu Y, Xu L. Design and synthesis of a gossypol derivative with improved antitumor activities. Arch Pharm (Weinheim). 2009 Apr;342(4):223-9.
[3] Arnold AA, Aboukameel A, Chen J, Yang D, Wang S, Al-Katib A, Mohammad RM. Preclinical studies of Apogossypolone: a new nonpeptidic pan small-molecule inhibitor of Bcl-2, Bcl-XL and Mcl-1 proteins in Follicular Small Cleaved Cell Lymphoma model. Mol Cancer. 2008 Feb 14;7:20.

Apogossypolone induces reactive oxygen species accumulation and controls cell cycle progression in Raji Burkkit's lymphoma cells.[Pubmed:25738577]

Mol Med Rep. 2015 Jul;12(1):337-44.

Burkitt's lymphoma (BL) is a highly aggressive type of non-Hodgkin's lymphoma, with marked rates of proliferation and metabolism. The expression levels of the translocated cellular Myc (c-Myc) oncogene and Epstein-Barr virus infection have an oncogenic role in facilitating tumor progression and maintaining a malignant phenotype in BL Raji cells. The present study identified that more reactive oxygen species (ROS) were produced in Raji cells compared with other types of malignant B cells. Cells exhibiting higher ROS levels suggested facilitation of the induction of cell death by ROS-induction compounds. In the present study, Apogossypolone (ApoG2) was observed to induce marked accumulation in the levels of ROS in the Raji cells, which damaged the cells and suppressed cell proliferation. Within 12 h following ApoG2 treatment, the Raji cells were prominently arrested in the G1 phase of the cell cycle. Immunoblotting analysis indicated that the chromodomain-helicase-DNA-binding protein 1, checkpoint kinase 1 and c-Myc proteins were significantly downregulated at 3, 6 and 12 h, respectively, following treatment. Following treatment with ApoG2 for 48 h, ApoG2 induced significant apoptosis in the Raji cells. This findings, together with our previous studies, which demonstrated ApoG2 as a potent inhibitor of anti-apoptotic B cell lymphoma 2 proteins, indicated that the ROS stimulatory effect of ApoG2 increased the antitumor activity of ApoG2.

Apogossypolone (ApoG2) induces ROS-dependent apoptosis and reduces invasiveness of PC12 cells in vitro and in vivo.[Pubmed:28979675]

Am J Transl Res. 2017 Sep 15;9(9):3990-4002. eCollection 2017.

Malignant pheochromocytoma is accurately diagnosed only at occurrence of metastatic foci. However, at that time, patients are less likely to get many benefits from traditional chemotherapy. Over-expression of BCL-2 family proteins is tightly correlated with progression of pheochromocytoma. ApoG2, as the most potent gossypol derivative, has exhibited anti-tumor activities in various tumors. In the present study, we found that the staining degree of Bcl-2 being stronger than Bax was more frequently observed in pheochromocytoma than adrenocorticohyperplasia, which was possibly related to shorter overall survival. In addition, ApoG2 could induce apoptosis through up-regulation of Bax and down-regulation of Bcl-2, increasing reactive oxygen species (ROS) levels, inducing cytochrome C release and cleaving caspase proteins. Most importantly, those inhibition effects were blocked by caspase activation inhibitor Z-VAD-fmk and antioxidant N-acetyl-L-cysteine. The above results were further confirmed in vivo. Furthermore, ApoG2 could effectively inhibit tumor movement capabilities. Altogether, our results indicated that ApoG2 was a potential effective target drug for pheochromocytoma.

ApoG2 inhibits the antiapoptotic protein, Mcl1, and induces mitochondriadependent apoptosis in human colorectal cancer cells.[Pubmed:26352605]

Mol Med Rep. 2015 Nov;12(5):6976-84.

Colorectal cancer (CRC) is a worldwide malignancy of high incidence and mortality. At present, there is a lack of effective drugs against CRC. The Bcell leukemia/lymphoma 2 (Bcl2) protein family members are considered to be closely associated with tumorigenesis and the chemoresistance of CRC. As a novel gossypol derivative targeting antiapoptotic proteins of the Bcl2 family, Apogossypolone (ApoG2) exhibits antitumor properties in various cancer types, although its effects against CRC remain to be fully elucidated. In the present study, the cytotoxicity of ApoG2 in vitro on CRC cells was investigated, with the aim of elucidating the underlying mechanism. Using an MTT assay, ApoG2 was revealed to inhibit the growth of the HT29, SW480 and HCT116 CRC cell lines in a dose and a timedependent manner. Hoechst staining revealed that ApoG2 induced CRC cell apoptosis, marked by morphological changes, including cell shrinkage and nuclear fragmentation. Flow cytometric analysis also detected a higher apoptotic ratio following treatment with ApoG2. The ratio was dependent upon the concentration of ApoG2, which the cells were exposed to, and the duration of the exposure. Western blot analysis and immunoprecipitation experiments revealed that ApoG2 treatment led to the downregulation of the protein expression of Mcl1, and the interruption of the binding of Mcl1 to the protein Bax. Furthermore, treatment with ApoG2 led to the release of cytochrome c into the cytoplasm and the activation of caspases 3 and 7. The present study revealed that ApoG2 inhibited the proliferation of the CRC cell lines through mitochondrial signaling pathwaydependent apoptosis, which may be associated with the disruption of the function of the Mcl1 protein by ApoG2.

Apogossypolone induces apoptosis and autophagy in nasopharyngeal carcinoma cells in an in vitro and in vivo study.[Pubmed:28693230]

Oncol Lett. 2017 Jul;14(1):751-757.

Nasopharyngeal carcinoma (NPC) has a high incidence and mortality rate, particularly in Southern China. Apogossypolone (ApoG2) is a novel derivative of gossypol with antitumor activity and less toxicity. The human NPC CNE-2 cell line was studied in the in vitro model; whilst 4 week-old male nude mice (BALB/c-nu) were inoculated subcutaneously with CNE-2 cells, and xenograft tumors were studied in the in vivo model. Graded concentrations of ApoG2 were used in treatment studies. In ApoG2-treated and control in vitro and in vivo tumor cells, cell apoptosis, and autophagy were evaluated and quantified using fluorescent and transmission electron microscopy and flow cytometry. Hoechst-33258 fluorescence staining was used to evaluate apoptosis in treated and non-treated cell culture and xenograft NPC cells. Western blotting was performed on lysed tumor cells using primary antibodies to B-cell lymphoma-2 (Bcl-2), beclin-1, and beta-actin, and flow cytometry results indicated cell apoptosis rates of 3.90+/-0.34 and 19.52+/-1.18% in the control and ApoG2-treated cells, respectively (F=485.294, P<0.001). Western blot analysis showed that ApoG2 significantly decreased expression of the Bcl-2 protein in CNE-2 cells, when compared with control cells (F=68.909, P=0.001) and flow cytometry showed cell autophagy rates of 0.92+/-3.10% of control cells compared with 28.24+/-7.35% of ApoG2-treated cells (F=31.035, P=0.003). ApoG2 treatment significantly increased beclin-1 protein expression in CNE-2 cells (F=497.906, P<0.001). ApoG2 treatment inhibited NPC xenograft tumor growth by 65.49% (P<0.05). In conclusion, these results support a role for ApoG2 in inhibiting the growth of human NPC cells by inducing apoptosis and autophagy. Future controlled clinical studies could be planned, to define safety, efficacy and dosing regimens for ApoG2 as a potential treatment for patients with NPC.

Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism.[Pubmed:27331811]

Int J Mol Sci. 2016 Jun 20;17(5). pii: ijms17060973.

There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach.