OBJECTIVE: To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible. METHODS: Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity. RESULTS: Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity. CONCLUSIONS Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.
Mice ; Male ; Animals ; Gossypol/toxicity* ; Testis ; Seminiferous Tubules ; Spermatids ; Spermatogenesis ; Adenosine Triphosphatases/pharmacology*

The biflavonoid isochamaejasmin is mainly distributed in the root of Stellera chamaejasme L. (Thymelaeaceae) that is used in traditional Chinese medicine (TCM) to treat tumors, tuberculosis, and psoriasis. Herein, isochamaejasmin was found to show similar bioactivity against Bcl-2 family proteins to the reference Bcl-2 ligand (-)-gossypol through 3D similarity search. It selectively bound to Bcl-xl and Mcl-1 with Ki values being 1.93 ± 0.13 μmol·L(-1) and 9.98 ± 0.21 μmol·L(-1), respectively. In addition, isochamaejasmin showed slight growth inhibitory activity against HL-60 with IC50 value being 50.40 ± 1.21 μmol·L(-1) and moderate growth inhibitory activity against K562 cells with IC50 value being 24.51 ± 1.62 μmol·L(-1). Furthermore, isochamaejasmin induced apoptosis of K562 cells by increasing the intracellular expression levels of proteins of the cleavage of caspase-9, caspase-3, and PARP which involved in the Bcl-2-induced apoptosis pathway. These results indicated that isochamaejasmin induces apoptosis in leukemia cells by inhibiting the activity of Bcl-2 family proteins, providing evidence for further studying the underlying anti-cancer mechanism of S. chamaejasme L.
Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Biflavonoids ; pharmacology ; therapeutic use ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Gossypol ; pharmacology ; HL-60 Cells ; Humans ; Inhibitory Concentration 50 ; K562 Cells ; Leukemia ; drug therapy ; metabolism ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Thymelaeaceae ; chemistry ; bcl-2-Associated X Protein ; metabolism

To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 micromol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (DeltaPsi(m)) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of DeltaPsi(m) in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/*drug effects ; Cell Line ; Cell Proliferation/*drug effects ; Dose-Response Relationship, Drug ; Gossypol/*analogs & derivatives/pharmacology ; Membrane Potential, Mitochondrial/*drug effects ; Mice ; Mice, Inbred BALB C ; Reactive Oxygen Species/*metabolism ; Signal Transduction/*drug effects

To investigate the effects of gossypol acetate on apoptosis in primary cultured cells from patients with acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) and its synergistic effect with dexamethasone. The apoptosis-inducing effect of gossypol acetate on primary cultured leukemia cells was analyzed by flow cytometry (FCM). The effect of gossypol acetate on survival rates of Raji cells and mononuclear cells (MNC) from normal bone marrow were evaluated by MTT assay. After co-treatment with gossypol acetate and dexamethasone, the apoptosis rate of Raji cells was detected by FCM. The results showed that gossypol acetate was able to induce apoptosis in primary cultured ALL cells at concentrations of ≥ 5 µmol/L. The effect was concentration and time dependent. Apoptosis-inducing concentration in CLL cells was higher than that in ALL cells. After exposing to 50 µmol/L gossypol acetate for 48 h, the apoptosis rate of ALL and CLL cells were (90.4 ± 6.2)% and (51.7 ± 10.3)% separately. No major growth inhibitory effect was observed in MNC from normal bone marrow when they were exposed to gossypol acetate at concentrations lower than 10 µmol/L. After exposing for 48 and 72 h, the IC(50) of gossypol acetate for MNC from normal bone marrow was 7.1 and 9.1 times as much as the IC(50) of Raji cells. Co-treatment with 10 µmol/L gossypol acetate and dexamethasone remarkably increased the apoptosis rate of Raji cells. It is concluded that the gossypol acetate has apoptosis-inducing activity in primary cultured leukemia cells from patients diagnosed as ALL and CLL in vitro. The inhibitory effect of gossypol acetate on MNC from normal bone marrow is less prominent than that on Raji cells. Co-treatment with gossypol acetate and dexamethasone notably amplified the pro-apoptosis activity of the latter in Raji cells.
Apoptosis ; drug effects ; Cell Line ; Dexamethasone ; pharmacology ; Gossypol ; analogs & derivatives ; pharmacology ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; pathology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; pathology ; Tumor Cells, Cultured

BACKGROUNDOur previous studies suggested that low-dose gossypol combined with steroid hormones has a reversible antifertility role in adult male rats, and the course of treatment was shorter than that of either gossypol or steroid hormones alone. This result suggested that low-dose gossypol and steroid hormones have a drug synergistic effect on antifertility. The aim of the study was to find the target organs of the antifertility synergistic effect of the combined regimen.

METHODSThirty-two adult male rats were divided into four groups randomly: group GH, rats were fed orally with gossypol acetic acid (GA, 12.5 mg×kg(-1)×d(-1)) and desogestrel (DSG, 0.125 mg×kg(-1)×d(-1))/ethinylestradiol (EE, 0.025 mg×kg(-1)×d(-1))/testosterone undecanoate (TU, 100 mg×kg(-1)×d(-1)); group G, a single dose of GA (12.5 mg×kg(-1)×d(-1)) was given; group H, the same dosage of DSG/EE/TU as in group GH were administered; group C, rats were treated with vehicle (1% methyl cellulose) as control. Testes and epididymis were removed at 8 weeks post-treatment for evaluating their weight, volumes, volume fraction, and total volume of testicular tissue structures and the seminiferous tubule diameter using stereological assay. Sperm cell numbers and the motility of epididymal sperm were quantitated by flow cytometry and morphological methods.

RESULTSCompared with group C, spermatogenesis was normal in group G and suppressed in groups H and GH. Similar changes of testicular tissue structures and sperm number were found in groups H and GH. The decreases of epididymal sperm number and motility in group GH were greater than that of the low-dose gossypol or steroid hormones alone group.

CONCLUSIONSThe suppression of spermatogenesis was induced by steroid hormones in the combined regimen, and the epididymis was the target organ of low-dose gossypol. Combined use of low-dose gossypol and steroid hormones played a comprehensive antifertility role in their synergistic effect on reducing the number and motility of epididymal sperm.

Animals ; Desogestrel ; pharmacology ; Epididymis ; drug effects ; Ethinyl Estradiol ; pharmacology ; Flow Cytometry ; Gossypol ; analogs & derivatives ; pharmacology ; Male ; Random Allocation ; Rats ; Sperm Motility ; drug effects ; Spermatogenesis ; drug effects ; Spermatozoa ; drug effects ; Testis ; drug effects ; Testosterone ; analogs & derivatives ; pharmacology

The present study was conducted to investigate the effects of gossypol acetic acid (GAA) on the proliferation of human mucoepidermoid carcinoma cell line MEC-1 in vitro and its possible molecular mechanisms of DNA double-strand breaks (DSB). MTT assay was performed to test the inhibition of proliferation of MEC-1 cells by GAA. DSB and γH2AX foci formation induced by GAA were detected by neutral comet assay and immunostaining. GAA (5-40 μmol/L) inhibited the growth of MEC-1 cells in a dose- and time-dependent manner. One of the indexes of comet assay, percentage of head DNA was decreased, however other indexes, including tail length, percentage of tail DNA, tail moment (TM) and Olive tail moment (OTM) were increased when treated with 2.5- 40 μmol/L GAA for 24 h or 20 μmol/L GAA for 3-48 h, compared with those in control. The percentage of γH2AX-positive cells was also increased when MEC-1 was treated with 2.5-20 μmol/L GAA for 24 h or 20 μmol/L GAA for 3-48 h, compared with that in control. All these results show that GAA inhibits the proliferation of MEC-1, and DSB maybe one of the mechanisms of inhibitory effect of GAA on the growth of tumor cells.
Antineoplastic Agents, Phytogenic ; pharmacology ; Carcinoma, Mucoepidermoid ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Breaks, Double-Stranded ; drug effects ; Gossypol ; analogs & derivatives ; pharmacology ; Humans ; Parotid Neoplasms ; genetics ; pathology

Apogossypolone (ApoG2), a novel derivative of gossypol, has been shown to be a potent inhibitor of antiapoptotic Bcl-2 family proteins and to have antitumor activity in multiple types of cancer cells. Recent reports suggest that gossypol stimulates the generation of cellular reactive oxygen species (ROS) in leukemia and colorectal carcinoma cells; however, gossypol-mediated cell death in leukemia cells was reported to be ROS-independent. This study was conducted to clarify the effect of ApoG2-induced ROS on mitochondria and cell viability, and to further evaluate its utility as a treatment for nasopharyngeal carcinoma (NPC). We tested the photocytotoxicity of ApoG2 to the poorly differentiated NPC cell line CNE-2 using the ROS-generating TL/10 illumination system. The rapid ApoG2-induced cell death was partially reversed by the antioxidant N-acetyl-L-cysteine (NAC), but the ApoG2-induced reduction of mitochondrial membrane potential (MMP) was not reversed by NAC. In the presence of TL/10 illumination, ApoG2 generated massive amounts of singlet oxygen and was more effective in inhibiting cell growth than in the absence of illumination. We also determined the influence of light on the anti-proliferative activity of ApoG2 using a CNE-2-xenograft mouse model. ApoG2 under TL/10 illumination healed tumor wounds and suppressed tumor growth more effectively than ApoG2 treatment alone. These results indicate that the ApoG2-induced CNE-2 cell death is partly ROS-dependent. ApoG2 may be used with photodynamic therapy (PDT) to treat NPC.
Animals ; Antineoplastic Agents ; pharmacology ; Cell Death ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gossypol ; analogs & derivatives ; pharmacology ; Humans ; Light ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Nude ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Transplantation ; Photochemotherapy ; Reactive Oxygen Species ; metabolism ; Singlet Oxygen ; metabolism ; Tumor Burden ; drug effects

This study is to investigate the influence and the expression of CMTM family of testosterone on spermatogenesis suppression in the male rats treated by gossypol and cyclophosphamide. Gossypol (50 mg kg(-1)) and cyclophosphamide (20 mg kg(-1)) were administered to male rats to induce spermatogenesis suppression. Testosterone propionate was administrated at the dose of 5 mg kg(-1) every other day for 6 times. Sperm was collected from the left caudal epididymis, the count and motility of sperm were analyzed by CASA. Morphological change of testis tissue was observed with HE staining. The expression of CMTM family was examined by Western blotting assay. Gossypol (50 mg kg(-1)) and cyclophosphamide (20 mg kg(-1)) decreased the count and motility of sperm, and the pathological change of testis tissue was also observed. But, testosterone (5 mg kg(-1)) had positive effect. Furthermore, CMTM4 down-expressed remarkably in the gossypol and cyclophosphamide treated rats, the expression of the CMTM4 was up-expressed after testosterone administration. On the contrary, the expression of CMTM2 increased significantly only in gossypol treated male rats, but not in cyclophosphamide treated male rats. The expression of CMTM2 was down-expressed after testosterone administration. However, no obvious change of CMTM2 was observed in cyclophosphamide treated rats. Testosterone did not influence the expression of CKLF1, CMTM3 and CMTM5, the CMTM6, CMTM7 and CMTM8 of CMTM family were not detected in testis tissue. These demonstrated that the spermatogenesis effect of testosterone (5 mg kg(-1)) was associated with the expression of CMTM family, and CMTM2 and CMTM4 may take part in the spermatogenesis process.
Animals ; Cyclophosphamide ; toxicity ; Gene Expression Regulation ; Gossypol ; toxicity ; Male ; Membrane Proteins ; metabolism ; Rats ; Rats, Wistar ; Sperm Count ; Sperm Motility ; drug effects ; Spermatogenesis ; drug effects ; Testis ; metabolism ; pathology ; Testosterone ; pharmacology

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.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gossypol ; analogs & derivatives ; pharmacology ; Humans ; Membrane Potential, Mitochondrial ; Multiple Myeloma ; pathology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the effects of gossypol acetate on proliferation and apoptosis in Raji lymphoblastoid cells and explore the possible mechanism.

METHODSTrypan blue staining and ethyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay were performed to measure the effect of gossypol acetate on the growth of Raji cells. The morphologic changes were observed with Wright's staining assay. Apoptosis was identified by agarose-gel electrophoresis and annexin V-FITC marked flow cytometry (FCM) analysis. The distribution of cell cycle, apoptosis rate, and Bcl-2 protein expression were analyzed by FCM. Caspase-3 activity was detected by colorimetric assay.

RESULTSGossypol acetate inhibited proliferation and induced apoptosis of Raji cells at concentration higher than 5 micromol/L. The effects were both dose- and time- dependent. Cycle analysis indicated the alteration of cell cycle and G0/G1 arrest. The activation of Caspase-3 was observed by colorimetric assay. The results of flow cytometry showed that the down-regulation of Bcl-2 protein expression and the activation of Caspase-3 seemed to occur simultaneously.

CONCLUSIONGossypol acetate can inhibit the growth of Raji cells and induce their apoptosis. The mechanism may be related to the alteration of cell cycle and the down-regulation of Bcl-2 protein expression.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gossypol ; analogs & derivatives ; pharmacology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism