Kaempferitrin induces apoptosis via intrinsic pathway in HeLa cells and exerts antitumor effects

Ethnopharmacological relevance

Justicia spicigera is used for the empirical treatment of cervical cancer in Mexico. Recently, we showed that Justicia spicigera extracts exerted cytotoxic and antitumoral effects and the major component of this extract was kaempferitrin (KM).

Materials and methods

The cytotoxic and apoptotic effect of KM on human cancer cells and human nontumorigenic cells were evaluated using MTT and TUNEL assays, and Annexin V/Propidium iodide detection by flow cytometry. The effect of KM on cell cycle was analyzed by flow cytometry with propidium iodide. The apoptotic and cell cycle effects were also evaluated by western blot analysis. Also, different doses of KM were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 32 days. The growth and weight of tumors were measured.

Results

KM induces high cytotoxic effects in vitro and in vivo against HeLa cells. The general mechanisms by which KM induces cytotoxic effects include: cell cycle arrest in G1 phase and apoptosis via intrinsic pathway in a caspase dependent pathway. Also, KM exerts chemopreventive and antitumor effects.

Conclusion

KM exerts cytotoxic and antitumor effects against HeLa cells.     

Cinobufacini,an aqueous extract from Bufo bufo gargarizans Cantor,induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells

Aim of the study

Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG₂ and Bel-7402.

Materials and methods

The effects of cinobufacini on cell proliferation of HepG₂ and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Δψm) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.

Results

Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Δψm) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).

Conclusion

The present study indicated that cinobufacini can induce apoptosis of HepG₂ and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.     

Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo

Ethnopharmacological relevance

Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action.

Materials and methods

The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice.

Results

Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo.

Conclusion

This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.     

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana

Ethnopharmacological relevance

Tridax procumbens is an active herb against leishmaniasis.

Aim of the study

Leishmaniasis is a group of diseases caused by Leishmania protozoa. We investigated the antileishmanial activity of Tridax procumbens extracts and a pure compound against promastigotes of Leishmania mexicana, the causative agent of cutaneous leishmaniasis in the New World.

Materials and methods

Extracts and (3S)-16,17-didehydrofalcarinol (1) were obtained by chromatographic methods from Tridax procumbens, and the latter identified by spectroscopic analysis. The effect of these extracts and 1 on the growth inhibition of promastigotes of Leishmania mexicana was evaluated. In order to test the safety of extracts and 1, mammalian cells were treated with them, and cell viability was assessed using trypan blue and MTT.

Results

We demonstrated that extracts of Tridax procumbens and 1 showed a pronounced activity against Leishmania mexicana. The methanol extract inhibited promastigotes growth of Leishmania mexicana with a 50% inhibitory concentration (IC₅₀) of 3 μg/ml, while oxylipin 1 exhibited the highest inhibition at IC₅₀ = 0.478 μg/ml.

Conclusions

In this study we report the biological activity of extracts and (3S)-16,17-didehydrofalcarinol (1), obtained from Tridax procumbens, on the promastigote form of Leishmania mexicana, with no effect upon mammalian cells.     

Standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal induces apoptosis via mitochondrial pathway in human cervical cancer HeLa cells

Ethnopharmacological relevance

Artemisia princeps Pampanini is widely used in Eastern traditional medicine for the treatment of circulatory disorders, such as, dysmenorrhea, hematuria, hemorrhoids, and inflammation, and is also used to treat chronic conditions, such as, cancers, ulcers, and digestive disorders.

Aim of the study

The purpose of this study is to investigate the effect of a standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal (FRAP) on the induction of apoptosis and the molecular mechanism involved in human cervical cancer HeLa cells.

Materials and methods

Human cervical cancer HeLa cells were treated with FRAP and apoptosis was detected by cell morphologic observation, annexin-V-PI staning and western blot analysis on the expression of protein associated with cell death.

Results

FRAP led to the cleavages of caspase-3, -8, and -9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD-fmk) significantly suppressed the FRAP-induced accumulation of annexin V positive cells. Furthermore, it was found that FRAP caused a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c to the cytosol. Furthermore, the overexpression of Bcl-xL significantly prevented FRAP-induced apoptosis, MMP changes, and the activations of caspase-3, -8, and -9. Interestingly, pretreatment with caspase-8 inhibitor significantly reduced the FRAP-induced activation of caspase-3 but not that of caspase-9, whereas the caspase-3 inhibitor, z-DEVD-fmk, markedly attenuated the FRAP-induced activation of caspase-8. In BALB/c^nu/nu mice bearing a HeLa xenograft, FRAP dosed at 25 or 50 mg/kg significantly inhibited tumor growth.

Conclusion

Our results indicate caspase-mediated activation of the mitochondrial death pathway plays a critical role in the FRAP-induced apoptosis of HeLa cells and that FRAP inhibits the in vivo tumor growth of HeLa xenograft mice.     

Celastrus orbiculatus extract induces mitochondrial-mediated apoptosis in human hepatocellular carcinoma cells

Objective

To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus (C. orbiculatus) extract in human hepatocellular carcinoma cells.

Methods

Human hepatocellular carcinoma cells (HCCLM6) were treated with C. orbiculatus extract (COE) at different nontoxic concentrations (10, 20, 40, 80, and 160 μg/mL). The effect of COE on HCCLM6 viability was examined using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis.

Results

COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner. Apoptosis was accompanied by increased Bax expression and decreased Bcl-2 expression. In addition, COE treatment led to the release of cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Furthermore, activation of extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulation of Akt phosphorylation was observed.

Conclusion

COE induces mitochondrial-mediated, caspase-dependent apoptosis in HCCLM6 cells, which might be attributed to the activation of mitogen-activated protein kinase (MAPK) and inhibition of Akt signaling pathways. These data suggest that COE may be a potential treatment for human hepatocellular carcinoma.     

Devil's Claw-a review of the ethnobotany, phytochemistry and biological activity of Harpagophytum procumbens

Ethnopharmacological relevance

Harpagophytum procumbens subps. procumbens (Burch.) DC. ex Meisn. (Pedaliaceae) is an important traditional medicine growing in the Kalahari region of southern Africa where it is consumed as a general health tonic and for treating diverse ailments including arthritis, pain, fever, ulcers and boils.

Aim of the review

To provide a comprehensive overview of the ethnobotany, phytochemistry and biological activity of H. procumbens and possibly make recommendations for further research.

Materials and methods

Peer-reviewed articles on H. procumbens were acquired on Scopus, ScienceDirect and SciFinder, there was no specific timeline set for the search. A focus group discussion was held with different communities in Botswana to further understand ethnobotanical uses of the plant.

Results

Harpogophytum procumbens is used for a wide variety of health conditions in the form of infusions, decoctions, tinctures, powders and extracts. In addition to the common local use for arthritis and pain, other ethnomedicinal uses include dyspepsia, fever, blood diseases, urinary tract infections, postpartum pain, sprains, sores, ulcers and boils. Scientific studies revealed that H. procumbens exhibits analgesic, anti-oxidant, anti-diabetic, anti-epileptic, antimicrobial and antimalarial activities amongst others. Iridoid glycosides and phenylpropanoid glycosides have been the focus of phytochemical investigations as the biological activity has been ascribed to the iridoid glycosides (such as harpagoside and harpagide), which are common in nature and are known to possess anti-inflammatory activity. In addition, it has been shown that the hydrolysed products of harpagoside and harpagide have more pronounced anti-inflammatory activity when compared to the unhydrolysed compounds. Harpagophytum zeyheri is a close taxonomic ally of H. procumbens but H. procumbens is the favoured species of commerce, and contains higher levels of the pharmacologically active constituents. The two are used interchangeably and H. procumbens raw material is often intentionally adulterated with H. zeyheri and this may impact on the efficacy of inadequately controlled health products. The main exporter of this highly commercialised plant is Namibia. In 2009 alone, Harpagophytum exports were worth approximately €1.06 million. The high demand for health products based on this plant has led to over-harvesting, raising concerns about sustainability. Although only the secondary tubers are utilised commercially, the whole plant is often destroyed during harvesting.

Conclusions

Harpagophytum procumbens is used to treat a wide range of ailments. Some of the ethnobotanical claims have been confirmed through in vitro studies, however, when the constituents deemed to be the biologically active compounds were isolated the efficacy was lower than that of the whole extract. This necessitates the use of a different approach where all the metabolites are considered using a robust method such as spectroscopy; the phytochemical data can then be superimposed on the biological activity. Furthermore, there is a need to develop rapid and efficient quality control methods for both raw materials and products because the orthodox methods in current use are time-consuming and labour intensive.     

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3

Aim of the study

Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated.

Materials and Methods

Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunoflurenscence method. Activity of caspase-3 enzyme was measured by spectrofluorometry.

Results

MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Florescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate.

Conclusions

Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.     

Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway

Aim of the study

Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa.

Materials and methods

Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used.

Results

CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants.

Conclusions

These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.     

Cytotoxic effects of Echinacea purpurea flower extracts and cichoric acid on human colon cancer cells through induction of apoptosis

Ethnopharmacological relevance

Echinacea is a top-selling herbal supplement that acts as immunostimulant. It has been used to treat common cold, coughs, bronchitis and upper respiratory infections. It is also a popular product used in anticancer therapy. The cytotoxic effects of Echinacea on cancer cells are still not clear. The aims of this study were to provide a preliminary validation of the effects of 50% aqueous ethanol extract of Echinacea purpurea flowers and its major compound, cichoric acid, on human colon cancer cells Caco-2 and HCT-116.

Materials and methods

The cytotoxic effects of Echinace flower extracts and cichoric acid on cell viability, telomerase activity, DNA fragmentation, β-catenin, caspase-9, and cleavage of poly-ADP-ribose polymerase (PARP) of human colon cancer cell were examined.

Results

The results showed a significant inhibition of proliferation in E. purpurea flower extract and cichoric acid in a dose- and time-dependent manner. Cichoric acid treatment decreased telomerase activity in HCT-116 cells. Moreover, cichoric acid effectively induced apoptosis in colon cancer cells, which were characterized by DNA fragmentation, activation of caspase-9, cleavage of PARP and downregulation of β-catenin.

Conclusions

Our data indicate that cichoric acid has a strong growth-inhibitory effect against colon cancer cells, presumably resulting from the reduced telomerase activity and the induction of apoptosis. The exact mechanism of action should still be determined in future studies. Overall, the effects of 50% aqueous ethanol extract of E. purpurea flowers and cichoric acid may have provided in vitro evidence for the use as chemotherapeutic agents.     

MAPK activation is necessary to the apoptotic death of KB cells induced by the essential oil isolated from Artemisia iwayomogi

Aim of the study

Artemisia iwayomogi is a perennial small herbal plant that has long been used as a chemopreventive agent in traditional Korean medicine. Previously, the purified essential oil was isolated from Artemisia iwayomogi, herein named AIEO, and found to contain the active components responsible for the chemopreventive potential of the herb.

Materials and methods

This study examined whether or not AIEO has potential chemopreventative effects against cancer using the human oral epidermoid carcinoma cell line, KB cells. The possible mechanism of AIEO-induced apoptosis was also examined.

Results

The results showed that AIEO induces the apoptotic death of KB cells, which is mediated by mitogen-activated protein kinases (MAPKs). In addition, AIEO not only induced an imbalance between the mitochondrial Bcl-2 and Bax levels with the concomitant release of Cytochrome c into the cytosol but also induced the activation of caspases and the cleavage of PARP. This induction was significantly suppressed by MAPK inhibitors. Moreover, pretreating the cells with each of the caspase or MAPK-specific inhibitors apparently inhibited AIEO-induced cytotoxicity of KB cells.

Conclusions

These results strongly suggest that AIEO might have cancer chemopreventive and therapeutic potential, which is closely related to its ability to activate the MAPK-mediated signaling pathways with the subsequent induction of a mitochondria- and caspase-dependent mechanism.     

Chemoprevention of colonic aberrant crypt foci by Gynura procumbens in rats

Ethnopharmacological relevance

Gynura procumbens is commonly used as a traditional medicinal plant in Malaysia for treatment of many diseases. To investigate the chemopreventive properties of Gynura procumbens on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats.

Methods

Five groups of adult male rats were used in this experiment. Normal/control group; the rats were injected subcutaneously with 15 mg/kg of sterile normal saline once a week for two weeks, and orally administered with 10% Tween 20 (5 mL/kg). Carcinogen and treatment groups; the rats were injected subcutaneously each with 15 mg/kg body weight AOM once a week for 2 weeks and were continued to be fed for two months, respectively with 10% Tween 20, 500 and 250 mg/kg body weight plant extracts. Reference group; the rats were injected subcutaneously with 15 mg/kg body weight AOM once a week for 2 weeks, and injected intraperitoneally with fluorouracil 35 mg/kg body weight for five consecutive days.

Result

Total ACF detected in methylene blue stained whole mounts of rat colon were 21, 23and 130 in rats fed with 500, 250 mg/kg body weight treatment and carcinogen groups, respectively. Treatment with high and low doses of the plant extract led to83.6% and 82.2% decrease in the total crypts in the groups fed 500 mg/kg and 250 mg/kg Gynura procumbens respectively compared to carcinogen group. Immunohistochemical staining of ACF showed suppressed azoxymethane induced colonic cell proliferation and Bcl-2 expression. Glutathione-S-transfarase and superoxide dismutase activities were higher in treated rats compared to carcinogen groups.

Conclusion

Gynura procumbens reduced the incidence of AOM induced ACF. The findings showed that Gynura procumbens may have antiproliferative and antioxidative properties. Moreover, Gynura procumbens possesses the medicinal properties to prevent colon cancer.     

Physalin F from Physalis minima L. triggers apoptosis-based cytotoxic mechanism in T-47D cells through the activation caspase-3- and c-myc-dependent pathways

Ethnopharmacological relevance

Physalin F (a secosteroid derivative), is well recognized as a potent anticancer compound from Physalis minima L., a plant that is traditionally used to treat cancer. However, the exact molecular anticancer mechanism remains to be elucidated.

Aim of the study

We have recently reported the apoptosis-based cytotoxic effect of the chloroform extract of this plant. Here, we investigated the cytotoxicity and possible cell death mechanism elicited by the active constituent, physalin F on human breast T-47D carcinoma.

Materials and methods

Cytotoxic-guided fractionation of the chloroform extract of Physalis minima has led to the isolation of physalin F. The cytotoxicity activity was assayed using MTS assay. The effect of the compound to induce apoptosis was determined by biochemical and morphological observations through DeadEnd Colorimetric and annexin V assays, respectively, and RT-PCR analysis of mRNA expression of the apoptotic-associated genes.

Results

Cytotoxicity screening of physalin F displayed a remarkable dose-dependant inhibitory effect on T-47D cells with lower EC₅₀ value (3.60 μg/ml) than the crude extract. mRNA expression analysis revealed the co-regulation of c-myc- and caspase-3-apoptotic genes in the treated cells with the peak expression at 9 and 12 h of treatment, respectively. This apoptotic mechanism is reconfirmed by DNA fragmentation and phosphatidylserine externalization.

Conclusion

These findings indicate that physalin F may potentially act as a chemopreventive and/or chemotherapeutic agent by triggering apoptosis mechanism via the activation of caspase-3 and c-myc pathways in T-47D cells.     

Total saponins of Panax Notoginseng modulate the expression of caspases and attenuate apoptosis in rats following focal cerebral ischemia-reperfusion

Ethnopharmacological relevance

Total saponins of Panax notoginseng (TSPN), main constituents extracted from Panax Notoginseng, a highly valued traditional Chinese medicine, have been shown to be an effective agent on cerebral infarction.

Aim of the study

The effects of TSPN on apoptosis and expressions of caspase-1, caspase-3 and caspase-8 were studied after cerebral ischemia for 2 h followed by reperfusion for 46 h in rats.

Materials and methods

Rats were subjected to transient middle cerebral artery occlusion (MCAO) model using the intraluminal thread. TSPN was administered intraperitoneally at 5 min before and 12 h, 24 h and 36 h after MCAO, respectively.

Results

TSPN (at the dose of 25 mg/kg) significantly attenuated TUNEL-positive cells and reduced the expression of caspase-1 and caspase-3 compared to the model group, while it had no obvious effect on the expression of caspase-8.

Conclusions

The neuroprotective effect of TSPN on focal ischemia may be related to inhibition of apoptosis and caspases activation.     

Kaempferitrin induces immunostimulatory effects in vitro

Ethnopharmacological relevance

Justicia spicigera is a plant used as immunostimulatory in Mexican traditional medicine. Recently, we showed that Justicia spicigera extracts exerted immunostimulatory effects and the major component of this extract was kaempferitrin (KM). This work shows a correlation between the medical traditional use of Justicia spicigera and kaempferitrin, its active compound.

Materials and methods

The in vitro immunostimulatory effects of KM were evaluated on the proliferation of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of KM on NO production, lysosomal enzyme activity and neutral red uptake were assayed in murine macrophages RAW 264.7. The effects of KM on the NK cell activity were also assayed.

Results

KM at 25 μM, the highest concentration tested, increased the proliferation of murine macrophages (23%) and splenocytes (17%), and human PBMC (24%) in the absence of lipopolysaccharides (LPS), compared to untreated cells. KM also stimulated the pinocytosis (25%) and lysosomal enzyme activity (57%) in murine macrophages with a similar potency than LPS 1 μg/ml. In addition, KM induced the NK cell activity (11%).

Conclusion

KM exerts immunostimulatory effects on immune responses mediated by splenocytes, macrophages, PBMC and NK cells.     

Total alkaloids of Tripterygium hypoglaucum (levl.) Hutch inhibits tumor growth both in vitro and in vivo

Ethnopharmacological relevance

Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH) root is a traditional Chinese medicinal herb commonly used for treating autoimmune diseases and cancer. Alkaloid is one of the most bioactive components of THH extract. To evaluate the in vitro and in vivo antitumor properties of the total alkaloids of THH (THHta).

Materials and methods

THHta was extracted in pilot-scale. HCT116 cells were chose to establish human colon cancer xenograft model. The in vitro anti-tumor activity of THHta was tested by Cell malignant transformation test, Soft agar colony formation assay and MTT assay. The in vivo anti-tumor effect of THHta was confirmed by xenograft mouse model. THHta-induced apoptosis was examined by flow cytometry. The levels of apoptosis-related proteins were investigated by Western blot.

Results

TPA-induced cell transformation was significantly inhibited by THHta in JB6 Cl41 cells. THHta inhibits the growth of colon cancer cells in vitro in a significant dose-dependent manner. Compared to the control set, i.p. administration of THHta to xenograft mice signi?cantly reduced both tumor weight and volume. Apoptosis induction of THHta was mediated by activation of caspase-3, PARP and inhibiting of Bcl-2, Bcl-xL and XIAP.

Conclusion

THHta was effective in inhibiting tumor growth both in vitro and in vivo at less toxic concentrations by inducing apoptosis which suggested it could be developed as a potential anticancer agent.     

Protective effect of 5-hydroxymethylfurfural derived from processed Fructus Corni on human hepatocyte LO2 injured by hydrogen peroxide and its mechanism

Aim of the study

The aim of the present study was to evaluate the putative protective effect of 5-hydroxymethylfurfural (5-HMF) derived from processed Fructus Corni on human hepatocyte cell line (LO2) injured by hydrogen peroxide in vitro and the mechanism of its protection.

Materials and methods

The percentage of cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The hepatocyte cell apoptosis and cell cycle were detected by flow cytometric analysis. The content of nitric oxide and caspase-3 activity were quantified spectrophotometrically by enzyme-linked immunoassay.

Result

The study showed that incubation with 5-HMF caused significant increase in the viability of LO2 cell, decrease of cell apoptosis and recovery of cell cycle in LO2 cell injured by hydrogen peroxide, which was accompanied with the decreased nitric oxide level and caspase-3 activity.

Conclusions

Our data suggest that 5-HMF protects LO2 cell against damage induced by hydrogen peroxide through inhibiting effect of cell apoptosis caused by promoting S phase to G2/M phase and the decreased caspase-3 activity and nitric oxide level. 5-HMF is one of the active principles in processed Fructus Corni.     

Monoterpene bisindole alkaloids,from the African medicinal plant Tabernaemontana elegans, induce apoptosis in HCT116 human colon carcinoma cells

Ethnopharmacological relevance

Tabernaemontana elegans is a medicinal plant used in African traditional medicine to treat several ailments including cancer. The aims of the present study were to identify anti-cancer compounds, namely apoptosis inducers, from Tabernaemontana elegans, and hence to validate its usage in traditional medicine.

Methods and materials

Six alkaloids, including four monomeric indole (1–3, and 6) and two bisindole (4 and 5) alkaloids, were isolated from the methanolic extract of Tabernaemontana elegans roots. The structures of these compounds were characterized by 1D and 2D NMR spectroscopic and mass spectrometric data. Compounds 1−6 along with compound 7, previously isolated from the leaves of the same species, were evaluated for in vitro cytotoxicity against HCT116 human colon carcinoma cells by the MTS metabolism assay. The cytotoxicity of the most promising compounds was corroborated by Guava-ViaCount flow cytometry assays. Selected compounds were next studied for apoptosis induction activity in HCT116 cells, by evaluation of nuclear morphology following Hoechst staining, and by caspase-3 like activity assays.

Results

Among the tested compounds (1−7), the bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were found to be cytotoxic to HCT116 cells at 20 µM, with compound 5 being more cytotoxic than the positive control 5-Fluorouracil (5-FU), at a similar dose. In fact, even at 0.5 µM, compound 5 was more potent than 5-FU. Compounds 4 and 5 induced characteristic patterns of apoptosis in HCT116 cancer cells including, cell shrinkage, condensation, fragmentation of the nucleus, blebbing of the plasma membrane and chromatin condensation. Further, general caspase-3-like activity was increased in cells exposed to compounds 4 and 5, corroborating the nuclear morphology evaluation assays.

Conclusions

Bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were characterized as potent apoptosis inducers in HCT116 human colon carcinoma cells and as possible lead/scaffolds for the development of anti-cancer drugs. This study substantiates the usage of Tabernaemontana elegans in traditional medicine to treat cancer.     

Anticancer potential of aqueous extract of alocasia macrorrhiza against hepatic cancer in vitro and in vivo

Ethnopharmacological relevance

Alocasia macrorrhiza has been used as a folk medicine for cancer treatment in the Southwest of China.

Aim of the study

The purpose of this study is to confirm the anticancer activity of aqueous extract of alocasia macrorrhiza against hepatic cancer and to elucidate its mechanism of action.

Materials and methods

Human normal liver cells and hepatocellular carcinoma cells were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, apoptotic DNA fragmentation, and cell cycle distribution in response to alocasia macrorrhiza extract. The mRNA and protein expressions of PPARγ, Cyclin D1, Rb, P21, Bax, Bcl-2 and caspase-3 were detected through RT-PCR and Western blotting; the tumor growth inhibition in vivo was tested by oral administration of the extract.

Results

Alocasia macrorrhiza aqueous extract exhibited proliferation inhibition and apoptosis effects on human hepatocellular carcinoma cells in vitro, inhibited hepatoma growth in vivo.

Conclusion

Alocasia macrorrhiza extract has potential cytotoxic and apoptotic effect on human hepatocellular carcinoma cells and inhibits hepatoma growth in vivo, its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G₀/G₁) arrest, apoptosis induction through up-regulation the mRNA and protein expressions of PPARγ, Rb, Bax and capase-3genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes.     

Swietenia macrophylla King induces mitochondrial-mediated apoptosis through p53 upregulation in HCT116 colorectal carcinoma cells

Ethnopharmacological relevance

Swietenia macrophylla King is a traditional herb used to treat various diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect but the potential mechanisms have not been clearly defined. The current study was to further investigate the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-proliferative effect and apoptosis in HCT116 colorectal carcinoma cell.

Materials and methods

Cell viability was evaluated in HCT116 cells by trypan blue exclusion assay. Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. The apoptotic gene and protein expression were determined by Real-time quantitative PCR (q-PCR) and immunofluorescence staining using flow cytometry, respectively.

Results

SMEAF significantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive oxygen species (ROS) production. Moreover, the significant increase in p53 was accompanied by a decrease murine double minute 2 (MDM2) expression. SMEAF significantly increased the expression of the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation.

Conclusion

These results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-mediated pathway via p53 activation.