Carvacrol induces mitochondria-mediated apoptosis via disruption of calcium homeostasis in human choriocarcinoma cells.

Carvacrol is a monoterpenoid phenol present in the oils of various plants including Origanum vulgare (oregano) or Origanum majorana (marjoram). For a long time, it has been used as spice in foods because of its antimicrobial properties. Additionally, it appears to have anticancer effects against some cancer but this has not been well studied. Therefore, we conducted various assays to confirm the effects of carvacrol on choriocarcinoma cell lines (JAR and JEG3). Our results indicate that carvacrol has antiproliferative properties and induces apoptosis, resulting in increased expression of proapoptotic proteins. Additionally, carvacrol disrupted the mitochondrial membrane potential and induced calcium ion overload in the mitochondrial matrix in both JAR and JEG3 cells. Furthermore, carvacrol generated oxidative stress and lipid peroxidation in both JAR and JEG3 cells. Moreover, carvacrol-suppressed phosphoinositide 3-kinase-protein kinase B and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) signal transduction whereas expression of phosphor-P38 and c-Jun N-terminal kinase MAPK was increased. Together, our results indicate that carvacrol may be a possible new therapeutic agent or supplement for the control of human choriocarcinomas.

Naringenin suppresses growth of human placental choriocarcinoma via reactive oxygen species-mediated P38 and JNK MAPK pathways.

BACKGROUND: Human placental choriocarcinoma is a gestational trophoblastic tumor with high rates of metastasis and reoccurrence. However, some patients with choriocarcinoma are chemoresistance to conventional chemotherapeutic agents. HYPOTHESIS: Naringenin increases apoptosis in human placental choriocarcinoma cells. METHODS: We investigated the effects of naringenin on proliferation and migration of JAR and JEG3 cells, and performed TUNEL and Annexin V/PI staining assays to examine apoptotic effects of naringenin on both cells. In addition, we studied the loss of mitochondrial membrane potential (MMP) and the production of mitochondrial reactive oxygen species (ROS) to determine the specific reason for apoptosis of choriocarcinoma cells being mediated via mitochondria. Consistent with the induction of production of ROS by naringenin in both choriocarcinoma cell lines, we investigated lipid peroxidation and glutathione levels in both JAR and JEG3 cells since both are affected by ROS. We next determined dose-dependent effects of naringenin and its pharmacological inhibitors on signal transduction pathways in JAR and JEG3 cells by western blot analyses. RESULTS: Naringenin reduced viability and migratory functions of both cell lines, and increased mitochondria related apoptosis induced by ROS and lipid peroxidation, decreased glutathione and decreased mitochondrial membrane potential MMP in a dose-dependent manner. We also determined naringenin activated phosphorylation of ERK1/2, P38, JNK and P70S6K in JAR and JEG3 cells in a dose-response manner. Although naringenin induced phosphorylation of AKT proteins in JAR cells, it suppressed phosphorylation of the protein in JEG3 cells. In addition, we confirmed the mechanism of naringenin-induced cell signaling by using a combination of naringenin and pharmacological inhibitors of the PI3K and MAPK pathways, as well as a ROS inhibitor in JAR and JEG3 cell lines. CONCLUSIONS: Collectively, results of this study indicate that naringenin is a potential therapeutic molecule with anti-cancer effects on choriocarcinoma cells by inducing generation of ROS and activation of the MAPK pathways.

Silibinin stimluates apoptosis by inducing generation of ROS and ER stress in human choriocarcinoma cells.

Silibinin is a flavonolignan extracted from seeds of milk thistles. Traditionally, it has been used as a therapeutic agent for liver disorders, and now it is well-known for its anti-cancer effects. However, studies on anti-cancer effects of silibinin on choriocarcinoma are very limited. Therefore, we performed proliferation and apoptosis assays to determine effects of silibinin on the viability of human choriocarcinoma (JAR and JEG3) cells. Our results showed that silibinin significantly inhibited proliferation and induced apoptosis in both JAR and JEG3 cells, and significantly increased reactive oxygen species (ROS) and lipid peroxidation. Moreover, silibinin disrupted mitochondrial function by inducing permeabilization of mitochondrial membrane potential and calcium ion efflux in JAR and JEG3 cells. Furthermore, silibinin-induced apoptosis in choriocarcinoma cells via AKT, mitogen-activated protein kinases (MAPK) and unfolded protein response (UPR) signal transduction. Collectively, our results suggest that silibinin is a novel therapeutic agent or dietary supplement for management of human placental choriocarcinomas.

Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3.

BACKGROUND/AIMS: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. METHODS: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. RESULTS: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. CONCLUSION: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

Purified vitexin compound 1 suppresses tumor growth and induces cell apoptosis in a mouse model of human choriocarcinoma.

OBJECTIVE: In our previous study, we had isolated a series of lignan compounds, termed vitexins, from the seed of Chinese herb Vitex negundo and found broad antitumor activities of these compounds in many cancer xenograft models and cell lines. This study was aimed to determine the antitumor effect of purified vitexin compound 1 (VB1) on choriocarcinoma in vitro and in vivo. MATERIALS AND METHODS: The severe combined immunodeficiency mouse model of choriocarcinoma was established to investigate the in vivo effect of VB1. Its effect on proliferation and apoptosis in JEG-3 cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay and flow cytometry, respectively. The expression of caspase-3, Bcl-2, and some molecules involved in the mammalian target of rapamycin (mTOR) signaling was detected by Western blot. RESULTS: Vitexin compound 1 significantly inhibited the growth of choriocarcinoma in severe combined immunodeficient mice and reduced the serum ß-human chorionic gonadotropin level. Vitexin compound 1 inhibited cell proliferation, induced apoptosis, and inhibited the mTOR signaling in JEG-3 cell line. CONCLUSION: Vitexin compound 1 could inhibit choriocarcinoma via inducing cell apoptosis and suppressing the mTOR pathway.

Effects of phytoestrogen extracts isolated from flax on hormone production of trophoblast tumour cells Jeg 3 and BeWo.

UNLABELLED: AIM AND SETTING: To test the effects of crude extracts from flax (Linum usitatissimum) on progesterone and estradiol and ERα and ß/PR production in choriocarcinoma cell lines Jeg 3 and BeWo. Tumor trophoblast cells (Jeg 3 and BeWo) were incubated in the presence of different concentrations of the flax crude extracts. Estradiol and progesterone production was measured. Estrogen receptor α and ß as well as progesterone receptor expressions were also assessed. RESULTS: In Jeg 3 cells, progesterone production was downregulated by flax root and leaves extract, while in BeWo cells only flax root extract did manage to downregulate progesterone production. ERß expression was significantly downregulated by flax root and flax leaves extract in both cell lines; on the contrary, ERα expression was increased by flax leaves extract in BeWo cells. PR expression was downregulated by flax leaves extract in Jeg 3 and by flax root extract in BeWo cells. CONCLUSION: Flax extracts derived from leaves and especially from roots can modify progesterone and possibly estradiol production, while at the same time they seem to alter ERß expression. Further studies on animal models and adequately designed retrospective epidemiological studies are imperative to clarify this role upon progesterone.

Effects of bufalin on the proliferation of human choriocarcinoma cells.

OBJECTIVE: Bufalin is a traditional Chinese medicine, and it induces apoptosis in some lines of human tumor cells. METHODS: We investigated the effect of bufalin in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of bufalin, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. RESULTS: An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that BeWo cells were sensitive to the growth inhibitory effect of bufalin. Cell cycle analysis indicated that exposure to bufalin decreased the proportion of cells in the synthesis phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and by the loss of mitochondrial transmembrane potential. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. CONCLUSIONS: These results suggest that bufalin may serve as a therapeutic agent for the treatment of choriocarcinoma.

Effects of elm bark extracts from Ulmus laevis on human chorion carcinoma cell lines.

PURPOSE: The potential of substances from elm bark extracts to affect cancer has been described in several studies. In this study, the anticancer effects of extracts from Ulmus laevis bark were tested in hormone-dependent gynecological tumours using human chorion carcinoma cell lines. METHODS: The molecular-chemical composition of the bark extract was analysed by pyrolysis-field ionisation mass spectrometry. The influence of the extracts was determined on cell vitality and cytotoxicity in the human chorion carcinoma cell lines Jeg3 and BeWo in comparison with primary trophoblast cells. RESULTS: The elm bark extract was mainly composed of triterpenes, phytosterols, free fatty acids and suberins with lower amounts of dilignols and lipids. The elm bark extract significantly inhibited the vitality of Jeg3 and BeWo cells but increased the vitality of primary trophoblast cells. CONCLUSIONS: Substances extracted from elm bark might have beneficial effects for the prevention of hormone-dependent tumours.

Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells.

We investigated the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human choriocarcinoma (BeWo) cells. Treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC(50) values of 2.01 and 1.19 microM for 7 and 24 h respectively, accompanied by downregulation of proliferating cell nuclear antigen. Examination of nuclear morphology revealed fragmentation and condensation indicating apoptosis. Increase in the generation of reactive oxygen species (ROS) that was reversed by addition of reduced glutathione suggested ROS involvement in the cytotoxicity of nimbolide. A decrease in Bcl-2/Bax ratio with increased expression of Apaf-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase provide compelling evidence that nimbolide-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that nimbolide has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.

Cytotoxic activity of bryonolic acid isolated from transformed hairy roots of Trichosanthes kirilowii var. japonica.

Bryonolic acid was isolated in high yield from transformed hairy root cultures of Trichosanthes kirilowii var. Japonica. Bryonolic acid exhibited cytotoxic activity to various tumor cells in vitro, independent of cell type. Normal cells such as rat hepatocytes are less sensitive to bryonolic acid. The appearance of a DNA ladder was detected in the bryonolic acid-treated HL-60RG cells, indicating that cell death triggered by bryonolic acid is due to apoptosis.

[A study of first and second line chemotherapies in gestational choriocarcinoma].

Twenty-eight patients with choriocarcinoma have received the three kinds of combination chemotherapy since 1983 at our department, i.e., MOA consisting of moderate dose methotrexate (MTX), actinomycin-D (Act-D) and vincristine, MEA (moderate dose MTX, Act-D and etoposide) and FA (high dose 5-Fluorouracil and Act-D). The clinical and laboratory data obtained in the 28 patients were summarized as follows; 1. The MOA regimen was administered to 4 patients primarily and to 2 secondarily. All of the 6 patients attained remission, but finally two (33.3%) developed relapse. 2. The MEA regimen was administered to 12 patients primarily and to 12 secondarily. Of the 24 patients, five (20.8%) were found to be resistant to the MEA regimen. Nineteen patients (79.2%) attained remission, but 2 (10.5%) developed recurrence. 3. The FA regimen was attempted in one patient primarily and in 6 secondarily. Although one patient died, the remaining 6 achieved remission and one relapse has been observed in the 6 cases. 4. By applying the above mentioned 3 combination chemotherapy regimens, the overall survival rate was pushed up from 64% to 90% in choriocarcinoma patients. 5. Three patients finally died of the disease but not from the side effects of the combination chemotherapies. The major adverse effects were alopecia, nausea, vomiting and myelosuppression. In particular, serious myelosuppression was caused by the MEA or FA regimen in 5-7% of all chemotherapy courses.

[The treatment of refractory choriocarcinoma].

The choriocarcinoma was one of the most curable malignant neoplasms. This is chiefly because the effective chemotherapy was introduced and the sensitivity of hCG was improved. But about 10-20% cases with choriocarcinoma were drug resistant or recurrent cases. And now, the treatment of these refractory choriocarcinomas were one of the most important clinical problems. We describe a new combination chemotherapy for these refractory choriocarcinomas in this paper. This high dose 5-FU + Act-D protocol was effective for drug resistant cases with MEA protocol.

Photodynamic therapy of choriocarcinoma transplanted to the hamster cheek pouch. I. Intraperitoneal photosensitization.

Human choriocarcinoma (JEG-3) cells were transplanted into the cheek pouch of hamsters and treated with photodynamic therapy. Twenty-four hours after intraperitoneal injection of the photosensitizer dihematoporphyrin ether (DHE), 20 tumors were illuminated with 100 J/cm2 of 630-nm light from an argon pumped dye laser. Contralateral tumors served as controls. Dihematoporphyrin ether alone had no effect on tumor growth, while laser light in the absence of DHE resulted in complete regression in 3 tumors (17%), and partial regression in 4 of 18 tumors (22%), possibly due to hyperthermia, P greater than 0.10. Using the combination of DHE plus light (photodynamic therapy) complete tumor regression was noted after a single treatment in 11 of 20 tumors (55%, mean tumor volume 279 mm3) and in 7 of 7 tumors (100%) after a second treatment. Two of 20 tumors were not retreated. Therefore, 18 of 20 tumors (90%) were grossly destroyed by one or two photodynamic treatments. Contralateral control tumors continued to grow to a median volume of 990 mm3 (chi 2 = 26.30, P less than 0.0001). Choriocarcinoma transplanted into the hamster cheek pouch is highly responsive to photodynamic therapy.

[New combination chemotherapy against drug-resistant choriocarcinoma].

The remission rates of 373 patients with trophoblastic diseases were analysed according to the change in therapeutic methods. The following results were obtained. 1) In patients in the "low risk" and nonmetastatic "high risk" group, the remission rate was 100%. On the other hand, in those in the metastatic "high risk" group, the remission rate was only 71.4%, even if triple chemotherapy with methotrexate (MTX), actinomycin D (Act-D) and cyclophosphamide (CPM) was given primarily or secondarily. 2) Thirty-two of the patients in the metastatic "high risk" group underwent primary triple chemotherapy. Eleven additional patients in the same group who had never responded to initial chemotherapy of a different regimen had triple chemotherapy. The overall remission rates were 65.6% (21/32) and 45.5% (5/11), respectively. 3) The remission rate in those with recurrence who underwent triple chemotherapy was 44.4% (4/9). But two of these patients had a re-relapse. 4) Six of the patients in the metastatic "high risk" group had MEA protocol with a moderate dose of MTX, Act-D and etoposide. Five of them achieved remission. 5) The side effects of MEA protocol were not so serious, although alopecia and severe anemia were found in 100% and 75.8% of the patients, respectively.

[Anticancer effect of VP16-213 (etoposide) on three choriocarcinoma cell lines].

A new anticancer drug, VP16-213 was studied in vitro to determine its effect upon 3H-thymidine uptake and the survival rate of choriocarcinoma cells in comparison with the effect of MTX and Act-D. Three human choriocarcinoma cell lines, BeWo, HCCM-5 and SCH were used. The results were as follows; Serum VP16-213 levels were 10.3 +/- 1.19 micrograms/ml and 1.35 +/- 0.38 micrograms/ml at one hour and 12 hours after the administration of 100mg of VP16-213, respectively. In those three cell lines, VP16-213 suppressed cellular 3H-thymidine uptake by 70% as compared with the control. On the sixth day after exposure to VP16-213, the survival rate of choriocarcinoma cells was less than 10% in each of the three cell lines. One of the most prominent pharmacodynamic characteristics of VP16-213 was a rapid influx and efflux mechanism as seen in MTX. From the above, the anticancer effect of VP16-213 upon those three cell lines was supposed to be equal to or more than those of MTX and Act-D.

Activity and distribution studies of etoposide and mitozolomide in vivo and in vitro against human choriocarcinoma cell lines.

The in vivo antitumor activity of etoposide and mitozolomide was assessed in nude mice bearing a xenograft (CC3) of human gestational choriocarcinoma. Both agents demonstrated, at best, marginal activity observed as a delay in tumour growth. This lack of sensitivity suggests that the CC3 xenograft is not a good model for selection of agents for clinical evaluation in gestational choriocarcinoma. Plasma and tissue concentrations of etoposide and mitozolomide were measured in nude mice. Drug concentrations found in tumour tissue were 60% and 30% of plasma levels for mitozolomide and etoposide respectively. Etoposide and mitozolomide activity was also evaluated in vitro with another choriocarcinoma cell line (JAR). Maximum cell-kill was achieved after exposure to etoposide 0.05-1 microgram/ml for 3-24 h. In vitro response to etoposide demonstrates the importance of exposure time in determining cytotoxicity. In contrast, mitozolomide at concentrations from 1-100 micrograms/ml did not have a marked effect against JAR after exposure for 3-24 h.

Selective killing of choriocarcinoma cells in vitro by trichosanthin, a plant protein purified from root tubers of the Chinese medicinal herb Trichosanthes kirilowii.

Trichosanthin is an abortifacient plant protein purified from the Chinese medicinal herb Tian-hua-fen, obtained from root tubers of Trichosanthes kirilowii (Cucurbitaceae). Recently, trichosanthin has also been used in the treatment of trophoblastic tumours, including hydatidiform mole, invasive mole and choriocarcinoma. We have examined the effects of trichosanthin on cultured cells and observed selective cytotoxic effects on choriocarcinoma cells. Mouse melanoma cells were also sensitive to its cytotoxic action. Cytotoxic effects on cultured cells were usually not prominent after 24 hr treatment. Continued incubation for 48 hr in control culture medium after treatment with trichosanthin enhances the cytotoxicity on cells. Our findings suggest that trichosanthin exerts specific cytotoxic activity towards trophoblastic cells.

Reevaluation of 5-fluorouracil as a single therapeutic agent for gestational trophoblastic neoplasms.

Large dosage of 5-fluorouracil given by slow intravenous infusion has proved to be very effective in the treatment of gestational trophoblastic neoplasms. From 1965 through 1975, 5-fluorouracil was used as a single chemotherapeutic agent in 173 cases of invasive mole and 139 cases of choriocarcinoma. Complete remission was achieved in 84.9% of cases of invasive mole and in 59.3% of cases of choriocarcinoma when 5-fluorouracil was used as the initial treatment. Seven recurrences with three deaths occurred during follow-up in 216 patients who had achieved complete remission, providing a recurrence rate of 3.2% and recurrence death rate of 1.4%. All of the survivors were followed up for more than 5 years and 85.6% for more than 10 years. Toxicity of 5-fluorouracil was milder and less frequent than that of 6-mercaptopurine or methotrexate. The toxic reaction specific to 5-fluorouracil was diarrhea, which can result in pseudomembranous colitis if improperly treated. One of the two toxic deaths was due to this complication.

Chemotherapy of refractory germ cell cancer with Etoposide.

Eighteen patients with advanced germ-cell cancer (12 primary gonadal, six extragonadal) that was refractory to vinblastine (V), cisplatin (P), and bleomycin (B) were treated with Etoposide (VP-16-213) and cisplatin +/- bleomycin sulfate +/- doxorubicin hydrochloride. All patients experienced nausea, vomiting, alopecia, and myelosuppression. There were no treatment-related deaths. Five (42%) of 12 patients with primary gonadal germ-cell cancer achieved a complete remission and are presently alive with no evidence of disease. None of the six patients with extragonadal germ-cell cancer achieved a complete response. Thirteen patients died 6.2 months (median) after starting Etoposide treatment. Etoposide-containing chemotherapy is useful in patients with primary gonadal germ-cell cancer. Alternative therapies are needed for patients with extragonadal germ cell cancer.