Cytotoxic Effects of Darinaparsin, a Novel Organic Arsenical, against Human Leukemia Cells.

To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T-cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G2/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G2/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G2/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer.

Enhanced Cytotoxic Effects of Arenite in Combination with Active Bufadienolide Compounds against Human Glioblastoma Cell Line U-87.

The cytotoxicity of a trivalent arsenic derivative (arsenite, AsIII) combined with arenobufagin or gamabufotalin was evaluated in human U-87 glioblastoma cells. Synergistic cytotoxicity with upregulated intracellular arsenic levels was observed, when treated with AsIII combined with arenobufagin instead of gamabufotalin. Apoptosis and the activation of caspase-9/-8/-3 were induced by AsIII and further strengthened by arenobufagin. The magnitude of increase in the activities of caspase-9/-3 was much greater than that of caspase-8, suggesting that the intrinsic pathway played a much more important role in the apoptosis. An increase in the number of necrotic cells, enhanced LDH leakage, and intensified G2/M phase arrest were observed. A remarkable increase in the expression level of γH2AX, a DNA damage marker, was induced by AsIII+arenobufagin. Concomitantly, the activation of autophagy was observed, suggesting that autophagic cell death associated with DNA damage was partially attributed to the cytotoxicity of AsIII+arenobufagin. Suppression of Notch signaling was confirmed in the combined regimen-treated cells, suggesting that inactivation of Jagged1/Notch signaling would probably contribute to the synergistic cytotoxic effect of AsIII+arenobufagin. Given that both AsIII and arenobufagin are capable of penetrating into the blood-brain barrier, our findings may provide fundamental insight into the clinical application of the combined regimen for glioblastoma.

Synthesis of 4,4-Disubstituted 3,4-Dihydropyrimidin-2(1H)-ones and -thiones, the Corresponding Products of Biginelli Reaction Using Ketone, and Their Antiproliferative Effect on HL-60 Cells.

An efficient synthetic method for novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6 was developed. The cyclocondensation reaction of O-methylisourea hemisulfate salt 11 with 8 gives a tautomeric mixture of dihydropyrimidines 12 and 13 following acidic hydrolysis of the cyclized products to produce 5 in high yields. Thionation reaction of 5 at the 2-position smoothly proceeds to give 2-thioxo derivatives 6. These compounds 5 and 6, corresponding to the products of a Biginelli-type reaction using urea or thiourea, a ketone and a 1,3-dicarbonyl compound, have long been inaccessible and hitherto unavailable for medicinal chemistry. These methods are invaluable for the synthesis of 5 and 6, which have been inaccessible by conventional methods. Therefore, the synthetic methods established in this study will expand the molecular diversity of their related derivatives. These compounds were also assessed for their antiproliferative effect on a human promyelocytic leukemia cell line, HL-60. Treatment of 10 µM 6b and 6d showed high inhibitory activity similarly to 1 µM all-trans retinoic acid (ATRA), indicating that the 2-thioxo group and length of two alkyl substituents at the 4-position are strongly related to activity.

The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression.

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase ZL, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells.

Effects of hot­water extracts from 26 herbs on α­glucosidase activity.

α­glucosidase is a key enzyme that plays a role in glucose absorption in the gastrointestinal tract, and the inhibition of its activity induces the prevention of postprandial hyperglycemia. Several α­glucosidase inhibitors have been used as medicines for type 2 diabetes, but a similar effect is observed in natural resources, including traditional herbs and their phytochemicals. To identify the presence of the α­glucosidase inhibitory activity in herbs, in which various functional effects have been known to occur, the present study investigated the effects of hot­water extracts of 26 types of herbs on α­glucosidase activity in an in vitro assay. The results indicated significant increases in the inhibition of α­glucosidase activity in 1,000 µg/ml olive (P<0.01), white willow (P<0.01) and red rooibos hot­water extracts. Furthermore, ≥50% inhibition of α­glucosidase activity was determined to be significant in 1,000 µg/ml coltsfoot, green tea and bearberry hot­water extracts. In addition, the effects of bearberry, green tea and coltsfoot hot­water extracts on α­glucosidase activity in vivo were evaluated according to the blood glucose levels (BGLs) in maltose and glucose load model rats. It was indicated that the administration of these three herb extracts significantly reduced the increasing BGLs after maltose loading until 0.5 h compared with the control group. However, only coltsfoot extract significantly reduced the increasing BGLs after glucose loading until 0.5 h compared with the control group. Thus, the present results may facilitate the understanding of a novel functionality in traditional herbs, which could be useful for the prevention of disease onset and progression, such as in hyperglycemia and type 2 diabetes.

JNK and Autophagy Independently Contributed to Cytotoxicity of Arsenite combined With Tetrandrine via Modulating Cell Cycle Progression in Human Breast Cancer Cells.

Novel therapeutic strategies for breast cancer are urgently needed due to the sustained development of drug resistance and tumor recurrence. Trivalent arsenic derivative (arsenite, AsIII) has been reported to induce cytotoxicity in breast cancer cells. We recently demonstrated that AsIII plus tetrandrine (Tetra), a Chinese plant-derived alkaloid, exerted potent antitumor activity against human breast cancer cells, however, the underlying mechanisms for their action have not been well defined. In order to provide fundamental insights for understanding the action of AsIII plus Tetra, the effects of the combined regimen on two breast cancer cell lines T47D and MDA-MB-231 were evaluated. Compared to T47D cells, MDA-MB-231 cells were much more susceptible to the synergistic cytotoxic effects of AsIII and Tetra. Besides the induction of apoptotic/necrotic cell death, S-phase arrest and autophagic cell death were also observed in MDA-MB-231 cells. Exposure of MDA-MB-231 cells to AsIII and Tetra caused the activation of MAPKs. Cytotoxicity of the combined regimen in MDA-MB-231 cell was significantly abrogated by SP600125, a potent c-Jun N-terminal kinase (JNK) inhibitor. However, similar abrogation was not caused by p38 and ERK inhibitors. The addition of either autophagy inhibitors (3-methyladenine or wortmannin) or SP600125 corrected the combined regimen-triggered S-phase arrest, whereas had little effect on the apoptosis/necrosis induction in the cells. Surprisingly, SP600125NC, a negative control for SP600125, significantly strengthened S-phase arrest and the cytotoxicity induced by the combined regimen. The addition of SP600125 did not alter autophagy induction. In conclusion, the cytotoxicity of AsIII combined with Tetra was attributed to the induction of S-phase arrest, apoptotic/necrotic and autophagic cell death. The enhanced cytotoxicity of the two drugs by SP600125NC might be explained by its capability to strengthen S-phase arrest. Our results suggested that JNK and autophagy independently contributed to the cytotoxicity via modulating cell cycle progression. The study further provides fundamental insights for the development of AsIII in combination with Tetra for patients with different types of breast cancer.

Chemopreventive and anticancer activity of flavonoids and its possibility for clinical use by combining with conventional chemotherapeutic agents.

Cancer is a diverse class of diseases characterized by uncontrolled cell growth with the potential to invade and spread to other parts of the body, and continues to be one of the leading causes of death worldwide. Conventional cancer treatment modalities include antitumor drugs, surgical resection, locally targeted therapies such as radiation therapy. Along with improved understanding of the molecular pathogenesis of various cancers, generation and the use of smart targeted anti-cancer drugs have been challenged. The need for novel therapeutic strategies remains paramount given the sustained development of drug resistance, tumor recurrence, and metastasis. Development of new strategies aimed at improving chemotherapy sensitivity and minimizing the adverse side effects is thus essential for obtaining satisfied therapeutic outcomes for patients and enhancing their quality of life. Emerging evidence has reported that many cancer patients use either herbs employed in complementary therapies or dietary agents that influence cellular signaling worldwide. Numerous components of edible plants, collectively termed phytochemicals that have beneficial effects for health, are being reported increasingly in the scientific literature. Of those, flavonoids have attracted much attention by virtue of its wide variety of biological functions including antioxidant, anti-inflammatory, and anticancer activity. In this review, we highlight the molecular mechanisms underlying its multiple pharmacological effects, especially focusing on cancer chemoprevention. We further discuss possible strategies to develop anticancer therapy by combining flavonoids nutraceuticals and conventional chemotherapeutic agents. We also highlight numerous pharmacokinetic challenges such as bioavailability, drug-drug interactions, which are still fundamental questions concerning its future clinical application.

Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine.

BACKGROUND: To provide novel insight into the development of new therapeutic strategies to combat breast cancer, differentiation-inducing activity of clinically achievable concentrations of arsenite (AsIII) and tetrandrine (Tetra) was investigated in breast cancer cell lines MDA-MB-231 and MCF-7. METHODS: Differentiation induction of cancer cells was analyzed by flow cytometer. Alterations of genes related to differentiation, and proliferation of human normal peripheral blood mononuclear cells (PBMCs) were analyzed using western blotting and cell viability assay, respectively. RESULTS: Exposure to Tetra alone or in combination with AsIII induced differentiation of both cells characterized by upregulation of ICAM-1, downregulation of Her2/neu. In comparison with MCF-7, the combination of lower concentrations of AsIII and Tetra induced differentiation of MDA-MB-231, indicating that MDA-MB-231 cells were highly susceptible to differentiation. The differentiation occurred in parallel with activation of Erk signaling pathway, and was abolished by PD98059, a potent Erk inhibitor. Consistent with in vitro experimental results, the upregulation of ICAM-1 and the activation of Erk signaling pathway were also observed in MDA-MB-231 breast tumors in xenograft mouse obtained from our previous study. No obvious proliferation inhibition of PBMCs was observed following the exposure to AsIII combined with Tetra at the concentrations capable of inducing differentiation of MDA-MB-231 cells. CONCLUSION: The Erk signaling pathway may be crucially involved in the differentiation induction of breast cancer cells in vitro and in vivo. Collectively, our results suggest that the combination can probably serve as promising candidates for the development of novel therapeutic approaches for different types of breast cancer.

Enhanced cytotoxic effects of arsenite in combination with anthocyanidin compound, delphinidin, against a human leukemia cell line, HL-60.

Among five major anthocyanin compounds, delphinidin exhibited the most potent and selective cytocidal effect against HL-60, a trivalent arsenic (As(III))-resistant cell line. Co-treatment with delphinidin and As(III) resulted in the reduction of IC50 value for As(III) from 11.2 to 1.5 µM, which was considered as clinically achieved concentrations of As(III). The combination treatment strongly preferred to selectively enhance the cytotoxicity of As(III) against HL-60 cells rather than human peripheral blood mononuclear cells. The induction of apoptosis as evidenced by the increase of sub-G1 cells, DNA fragmentation, annexin V-positive cells and the activation of caspase-8, -9 and -3 was observed in HL-60 cells co-treated with As(III) and delphinidin. Similar to the activation pattern of caspases, a substantial decrease in the expression level of Bid along with the loss of mitochondrial membrane potential was also observed. These results suggested that the combination treatment triggered a convergence of the intrinsic and extrinsic pathways of apoptosis via the activation of caspase-8 and cleaved Bid. Delphinidin itself significantly decreased the intracellular GSH ([i]GSH) and nuclear factor-κB (NF-κB) binding activity, and further returned As(III)-triggered increment of [i]GSH and enhancement of NF-κB binding activity to control level. Additionally, buthionine sulfoximine, a GSH depletor; JSH-23, a NF-κB inhibitor, also mimicked the capacity of delphinidin to significantly induce the reduction of [i]GSH along with the potentiation of As(III) cytotoxicity in HL-60 cells. These observations suggested that delphinidin-induced sensitization of HL-60 cells to As(III) was caused by the reduction of [i]GSH, which was probably associated with the inhibitory effect of delphinidin on NF-κB binding activity. These findings further suggest that delphinidin-induced sensitization of HL-60 cells to As(III) may lead to dose reduction of As(III) in clinical application, and ultimately contribute to minimizing its side effects.

Rosehip inhibits xanthine oxidase activity and reduces serum urate levels in a mouse model of hyperuricemia.

Rosehip, the fruit of Rosa canina L., has traditionally been used to treat urate metabolism disorders; however, its effects on such disorders have not been characterized in detail. Therefore, the present study investigated the effects of hot water, ethanol and ethyl acetate extracts of rosehip on xanthine oxidase (XO) activity in vitro. In addition, the serum urate lowering effects of the rosehip hot water extract in a mouse model of hyperuricemia (male ddY mice, which were intraperitoneally injected with potassium oxonate) were investigated. Furthermore, the influence of rosehip hot water extract on CYP3A4 activity, which is the most important drug-metabolizing enzyme from a herb-drug interaction perspective, was investigated. Rosehip extracts of hot water, ethanol and ethyl acetate inhibited XO activity [half maximal inhibitory concentration (IC50) values: 259.6±50.6, 242.5±46.2 and 1,462.8±544.2 µg/ml, respectively]. Furthermore, the administration of 1X rosehip hot water extract significantly reduced the levels of serum urate at 8 h, which was similar when compared with the administration of 1 mg/kg allopurinol. Rosehip hot water extract only marginally affected CYP3A4 activity (IC50 value, >1 mg/ml). These findings indicate that rosehip hot water extract may present as a functional food for individuals with a high urate level, and as a therapeutic reagent for hyperuricemic patients.

Effects of tomato juice on the pharmacokinetics of CYP3A4-substrate drugs.

We previously demonstrated that tomato juice (TJ) contains potent mechanism-based inhibitor(s) of CYP3A4. In this study, we investigated the effects of TJ and grapefruit juice (GFJ) on the pharmacokinetics of the CYP3A4-substrate drugs, nifedipine (NFP) and midazolam (MDZ), in male Wistar rats. Oral administration of GFJ 90 min before the intraduodenal administration of NFP or MDZ increased the area under the concentration-time curves (AUCs) of NFP and MDZ by 32.4% and 89.4%, respectively. TJ increased MDZ blood concentrations and AUC after intraduodenal MDZ administration; however, it had no effect on NFP. When MDZ and NFP were intravenously administered, GFJ significantly increased the AUC of MDZ, but only slightly increased that of NFP. In contrast, TJ only slightly increased the AUC of MDZ. These results suggest that, similar to GFJ, TJ influences the pharmacokinetics of CYP3A4-substrate drugs; however, it may be a drug-dependent partial effect.

Cytotoxic effects of pyrrolidine dithiocarbamate in small-cell lung cancer cells, alone and in combination with cisplatin.

The cytocidal effect of pyrrolidine dithiocarbamate (PDTC) was investigated by focusing on cell viability, cell cycle arrest and apoptosis induction in small-cell lung cancer (SCLC) cell lines (NCI-H196 and NCI-H889). PDTC exhibited a much stronger dose-dependent cytotoxic activity against NCI-H196 compared to NCI-H889, while no such activity was observed in normal human embryonal lung fibroblast MRC-5 cells. Cell cycle arrest in S phase paralleled with suppression of c-myc expression without accompanying DNA fragmentation was observed in NCI-H196 cells. A transient increase in the intracellular ROS accompanied with an alteration of expression of oxidative stress-related genes was also confirmed in NCI-H196 cells. Furthermore, the addition of N-acetyl-l-cysteine (NAC), a free radical scavenger, not only abolished PDTC-trigger alterations of expression of these oxidative-related genes, but also almost completely abrogated PDTC-induced reduction in cell viability and morphological changes associated with cell damage. These results thus suggest that PDTC-induced cytotoxicity is attributed to its pro-oxidant activity. PDTC-induced cytotoxicity was further enhanced by CuCl2, however, abolished by bathocuproine disulfonate (BCPS), a non-permeable copper-specific chelator, supporting the plausibility that accumulation of intracellular Cu plays an important role in the cytotoxicity. Importantly, we demonstrated for the first time that PDTC downregulated the expression of ATP7A, known to be responsible for Cu efflux, but did not affect the expression of CTR1, known as a copper uptake transporter. Intriguingly, combination of much lower dose of cisplatin (5 µM) and non-toxic dose of PDTC (0.1 µM) synergistically induced a significant cytotoxicity in NCI-H196 cells. Given that ATP7A plays a critical role in the resistance of platinum-drug (such as cisplatin) representing a first-line treatment for SCLC, PDTC could be a promising candidate of adjunct therapeutic reagent for the patients requiring treatment with platinum-based regimens.

Involvement of histone H3 phosphorylation via the activation of p38 MAPK pathway and intracellular redox status in cytotoxicity of HL-60 cells induced by Vitex agnus-castus fruit extract.

We have demonstrated that an extract from the ripe fruit of Vitex angus-castus (Vitex), might be a promising anticancer candidate. In order to further provide a molecular rationale for clinical development in anticancer therapy, a detailed mechanism underlying the efficacy of Vitex against HL-60 cells was investigated. Vitex induced a dose- and time-dependent decrease in cell viability associated with induction of apoptosis and G(2)/M cell cycle arrest, both of which were suppressed by the addition of SB203580, an inhibitor for p38 MAPK. Furthermore, SB203580 significantly suppressed Vitex-induced phosphorylation of histone H3, a downstream molecule of p38 MAPK known to be involved in apoptosis induction in tumor cells. Notably, Vitex induced upregulation of intracellular ATP, known to bind its binding pocket inside activated p38 MAPK and to be required for the activation of p38 MAPK pathway. These results, thus, suggest that upregulation of intracellular ATP and phosphorylation of histone H3 are closely associated with the activation of p38 MAPK pathway, consequently contributing to Vitex-mediated cytotoxicity. Intriguingly, a significant decrease of intracellular ROS levels and downregulation of expression level of gp91(phox), an important component of NADPH oxidase, were observed in Vitex-treated cells. A greater decline in ROS levels along with enhanced apoptosis was observed after treatment with Vitex in combination with SnPP, an inhibitor specific for HO-1. Since NADPH oxidase and HO-1 are closely correlated to redox status associated with intracellular ROS levels, the two enzymes are suggested to be implicated in Vitex-mediated cytotoxicity in HL-60 cells by regulating ROS generation. We also suggest that activation of the p38 MAPK pathway may be dependent on the alterations of intracellular ATP levels, rather than that of intracellular ROS levels. These results may have important implications for appropriate clinical uses of Vitex and provide novel insights into the interaction between Vitex and other conventional drugs capable of affecting intracellular redox status.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D3 (VD3)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Involvement of histone H3 phosphorylation through p38 MAPK pathway activation in casticin-induced cytocidal effects against the human promyelocytic cell line HL-60.

The effect of casticin was investigated by focusing on cell viability, apoptosis induction and cell cycle arrest in HL-60 cells. Casticin induced a dose- and time-dependent decrease in cell viability associated with apoptosis induction and G2/M cell cycle arrest. The addition of SB203580, an inhibitor for p38 mitogen-activated protein kinase (MAPK), but not SP600125 [c-Jun NH2-terminal protein kinase (JNK) inhibitor] and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor], abrogated casticin-induced cell cycle arrest and apoptosis associated with the activation of caspases including caspase-8, -9 and -3. Endogenous p38 MAPK activation was observed in untreated cells based on the detection of the expression levels of phospho-p38 MAPK, whereas casticin did not affect the degree of p38 MAPK activation. Interestingly, the addition of SB203580 suppressed casticin-induced phosphorylation of histone H3, a downstream molecule of the p38 MAPK signaling pathway and known to be involved in chromosome condensation during mitosis. More importantly, casticin induced upregulation of intracellular ATP levels. Although casticin induced intracellular reactive oxygen species, antioxidants failed to block casticin-mediated cytotoxicity, indicating that casticin-induced cytotoxicity appears to be independent of reactive oxygen species generation. Based on the fact that SB203580 has been reported to compete with ATP for binding to the active form of p38 MAPK, and consequently blocks the p38 MAPK activity in activating downstream molecules, these results suggest that casticin induces cytotoxicity associated with apoptosis and cell cycle arrest in HL-60 cells through the p38 MAPK pathway, in which intracellular ATP levels and phosphorylation of histone H3 play critical roles.

Contribution of membrane progesterone receptor α to the induction of progesterone-mediated apoptosis associated with mitochondrial membrane disruption and caspase cascade activation in Jurkat cell lines.

In the present study, we investigated the effects of progesterone (Pg) on the growth of A3 and its caspase-8-deficient mutant cell line, I9.2, both of which are subclones of a T-cell-derived leukemic Jurkat cell line that lacks the classic cytoplasmic/nuclear Pg receptor. Pg inhibited the cell growth of both cell lines in a dose- and time-dependent manner to a similar extent, regardless of the status of caspase-8 expression. The activation of caspase-9 and -3 was observed in both cell lines following treatment with 50 µM Pg for 24 h. In addition, the activation of caspase-8 was observed in A3 cells. The addition of the pan-caspase inhibitor Boc-D-FMK, significantly suppressed Pg-triggered cytocidal effects in both types of cells. Moreover, exposure to 50 µM Pg for 48 and 72 h resulted in lactate dehydrogenase leakage characteristic of the disruption of cellular membrane integrity. The function of membrane progesterone receptor α coupled directly with the Gi protein was revealed based on the restoration of Pg-triggered loss of mitochondrial membrane potential in the presence of pertussis toxin, an inhibitor specific for Gi protein. These results suggest that growth suppression accompanied with induction of apoptosis by Pg in both Jurkat clone cells was mediated through mitochondrial membrane disruption followed by the activation of the caspase cascade, as a result of the activation of non-genomic effects. The results of the present study provide novel insight into Pg actions toward its use for clinical application in patients with lymphocytic T cell leukemia.

Cytotoxic effects of flavonoids against a human colon cancer derived cell line, COLO 201: a potential natural anti-cancer substance.

The proliferation of a human colon carcinoma cell line, COLO 201, was effectively suppressed through apoptosis in the presence of flavonoids, an ethanol extract from Vitex agnus-castus fruits. The induction of apoptosis was not inhibited by the presence of an anti-oxidant, N-acetyl-L-cysteine, whereas only HO-1 gene expression levels increased among other typical oxidative stress-associated genes examined after Vitex treatment. These results suggest that Vitex treatment activates a pathway associated with HO-1 gene activation, resulting in the induction of apoptosis in COLO 201. Results also implicate a potential clinical chemotherapeutic application of Vitex for the treatment of colon cancer patients.