Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells

Sutherlandia frutescens is a well-known South African herbal remedy traditionally used for stomach problems, internal cancers, diabetes, various inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The influence of crude Sutherlandia frutescens extracts (prepared with 70% ethanol) was investigated on cell numbers, morphology, and gene expression profiles in a MCF-7 human breast adenocarcinoma cell line. Time-dependent (24, 34, 48 and 72 h) and dose-dependent (0.5-2.5 mg/ml) studies were conducted utilizing spectrophotometrical analysis with crystal violet as DNA stain. A statistically significant decrease to 50% of malignant cell numbers was observed after 24 h of exposure to 1.5 mg/ml Sutherlandia frutescens extract when compared to vehicle-treated controls. Morphological characteristics of apoptosis including cytoplasmic shrinking, membrane blebbing and apoptotic bodies were observed after 24h of exposure. A preliminary global gene expression profile was obtained by means of microarray analysis and revealed valuable information about the molecular mechanisms and signal transduction associated with 70% ethanolic Sutherlandia frutescens extracts.     

Anti-diabetic effects of Sutherlandia frutescens in Wistar rats fed a diabetogenic diet

Sutherlandia frutescens has been marked as a potential hypoglycaemic agent for the treatment of type 2 diabetes. We investigated the effects of Sutherlandia frutescens in bringing about hypoglycaemia and promoting glucose uptake in pre-diabetic rats. Crushed Sutherlandia frutescens leaves in drinking water were administered to rats fed a high fat diet. Positive control rats received only metformin. Glucose uptake experiments were undertaken using [(3)H] deoxy-glucose. Various physiological parameters were also measured. Rats receiving Sutherlandia frutescens displayed normoinsulinaemic levels, after 8 weeks medicational compliance, compared to the fatty controls. There was a significant increase in glucose uptake into muscle and adipose tissue, and a significant decrease in intestinal glucose uptake (p<0.001 at 60min) in rats receiving the plant extract. The Sutherlandia frutescens plant extract shows promise as a type 2 anti-diabetes medication because of its ability to normalize insulin levels and glucose uptake in peripheral tissues and suppress intestinal glucose uptake, with no weight gain noted. The exact mechanism of action and the extract's efficacy in humans need further confirmation.     

中药对肝癌细胞信号转导通路影响的研究进展

随着对肿瘤信号转导通路研究的不断深入,人们对肿瘤细胞内部复杂的信号转导机制以及它们对肿瘤生长、凋亡和转移等的影响越来越了解.目前,中药及其提取物通过作用于信号转导通路诱导肝癌细胞凋亡、影响肝癌血管生成的研究已取得可喜进步.现对近年来有关文献进行回顾,就此进展作一综述.     

The apoptosis inducing effects of Sutherlandia spp. extracts on an oesophageal cancer cell line

Aim of study

Oesophageal cancer is the ninth most common cancer in the world and the second most common cancer among South African men. It also has one of the lowest possibilities of cure, with the 5-year survival rate estimated to be only 10% overall. Sutherlandia frutescens, or the “cancer bush”, is a medicinal plant indigenous to southern Africa that is believed to have anti-cancer and anti-proliferative properties. The aim of this study was to investigate the potential apoptosis-inducing effects of two S. frutescens extracts and one Sutherlandia tomentosa extract on the SNO oesophageal cancer cell line.

Materials and methods

Cell viability and morphology of SNO cells were evaluated following exposure to the extracts. Apoptotic markers including cytochrome c translocation and phosphatidylserine externalisation were quantified by flow cytometry. The activity of caspases 3 and 7 was evaluated with spectrofluorometry. Apoptosis was evaluated in the presence of the pan-caspase inhibitor, Z-VAD-fmk. The effect of the extracts was compared to non-cancerous peripheral blood mononuclear cells (PBMCs).

Results

Time- and dose-response studies were conducted to establish treatment conditions of 2.5 and 5 mg/ml of crude plant extracts. Microscopy studies revealed that S. frutescens- and S. tomentosa-treated SNO cells had morphological features characteristic of apoptosis. Annexin V/propidium iodide flow cytometry confirmed that the extracts do, in fact, induce apoptosis in the SNO cells. Caspase inhibition studies seem to indicate that extracts A (S. frutescens (L.) R. Br. subsp. microphylla from Colesberg), B (S. frutescens (L.) R. Br. subsp. microphylla from Platvlei) and C (S. tomentosa Eckl. & Zeyh from Stil Bay) are able to induce caspase-dependent as well as -independent cell death. The S. frutescens and S. tomentosa extracts were found to be more cytotoxic to cancerous SNO cells when compared to the PBMCs.

Conclusions

S. frutescens and S. tomentosa extracts show promise as apoptosis-inducing anti-cancer agents.     

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.     

Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks

Ethnopharmacological relevance

The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation.

Aim of the study

There is an increasing interest among researchers in searching for new anticancer drugs from natural resources, particularly plants. This study aimed to investigate the anticancer properties of Acalypha wilkesiana extracts and the characteristics of DNA damage against brain and lung cancer cells.

Materials and methods

The antiproliferative activity of Acalypha wilkesiana extracts (ethyl acetate, hexane, and ethanol) was examined on human glioma (U87MG), human lung carcinoma (A549), and human lung fibroblast (MRC5) cells.

Results

Cell viability MTT assay revealed that ethyl acetate extract of the plant possessed significant antiproliferative effects against both U87MG (GI₅₀ = 28.03 ± 6.44 μg/ml) and A549 (GI₅₀ = 89.63 ± 2.12 μg/ml) cells (p value < 0.0001). The hexane extract was found to exhibit crucial antiproliferative effects on U87MG (GI₅₀ = 166.30 ± 30.50 μg/ml) (p value < 0.0001) but not on A549 cells. Neither plant extract possessed noticeable antiproliferative effects on the non-cancerous MRC5 cells (GI₅₀ > 300 μg/ml). The ethanol extract showed no antiproliferative effects on any cell line examined. Haematoxylin &; Eosin (H &; E) staining and single cell gel electrophoresis (SCGE) comet assay confirmed that plant extract-treated cells underwent apoptosis and not necrosis. SCGE comet assays confirmed that plant extracts caused both single strand (SSB) and double strand (DSB) DNA breaks that led to the execution of apoptosis.

Conclusion

The extracts (especially ethyl acetate and hexane) of Acalypha wilkesiana possess valuable cytotoxic effects that trigger apoptosis in U87MG and A549 cancer cells through induction of DNA SSBs and DSBs.     

Effect of Perilla frutescens on nitric oxide production and DNA synthesis in cultured murine vascular smooth muscle cells

The effects of perilla (Perilla frutescens, Labiatae) on murine cultured vascular smooth muscle cells (VSMC) were investigated. The water extract of perilla leaves induced nitric oxide (NO) production of VSMC and this effect was synergistically augmented when combined with interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha, while the perilla extract significantly inhibited NO production induced by IFN-gamma combined with lipopolysaccharide (LPS). Northern blot analysis revealed that these effects of the perilla extract paralleled mRNA expression of inducible nitric oxide synthase. However, the perilla extract significantly inhibited platelet derived growth factor (PDGF) or TNF-alpha-induced VSMC proliferation measured as DNA synthesis. The inhibitory effect of the perilla extract on TNF-alpha-induced VSMC proliferation was significantly suppressed by N(G)-monomethyl-L-arginine, a non-specific nitric synthase inhibitor, suggesting that this effect was partially mediated by NO production as an autocrine/paracrine factor. The present findings suggest that perilla would be useful for the prevention of vascular diseases such as arteriosclerosis.     

Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells

Aim of the study

Pregnane glycosides are potent cytotoxic agents which may represent new leads in the development of anti-tumour drugs, particularly in the treatment of breast cancer, because of the structural similarity to estrogenic agonists. Caralluma species are natural sources of a wide variety of pregnane glycosides. The aim of the study was to isolate, using an activity-guided fractionation approach, novel pregnane glycosides for testing on breast cancer and other tumour lines.

Materials and methods

The effect of crude extracts, specific organic fractions and isolated compounds from Caralluma tuberculata was tested on the growth and viability of MCF-7 estrogen-dependent, and MDA-MB-468 estrogen-independent breast cancer cells, Caco-2 human colonic cells, HUVECs and U937 cells. Neutral red uptake and MTT assays were used. Apoptosis was detected by Western blot of poly-(ADP ribose) polymerase (PARP) as were other markers of nuclear fragmentation (DNA ladder assay, staining of cells with nuclear dye DAPI). The involvement of caspases was investigated using the pan-caspase inhibitor Z-VAD-FMK.

Results

The ethyl acetate fraction of Caralluma tuberculata was found to be the most potent anti-proliferative fraction against all three cancer cell lines. Two novel steroidal glycosides were isolated from the active fraction after a series of chromatographic experiments. The structure of the isolated compounds was elucidated solely based on 2D-NMR (HMBC, HETCOR, DQF-COSY) and MS spectral analysis as compound 1: 12-O-benzoyl-20-O-acetyl-3β,12β,14β,20β-tetrahydroxy-pregnan-3-ylO-β-D-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-3-methoxy-β-d-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3β,7β,12β,14β-tetrahydroxy-17β-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-β-d-glucopyranosyl-(1 → 4)-6-deoxy-β-d-allopyranosyl-(1 → 4)-β-d-cymaropyranosyl-(1 → 4)-β-d-cymapyranosyl-(1 → 4)-β-d-cymaropyranoside. Compound 1 (pregnane glycoside) and compound 2 (androstan glycoside) induced apoptosis at <25 μM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor.

Conclusions

Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design.     

Growth inhibitory and apoptosis inducing effect of Perilla frutescens extract on human hepatoma HepG2 cells

Perilla frutescens (L.) Britt. (Lamiaceae) has traditionally been used to treat diseases, including tumors, but the antitumorigenesis mechanism is unclear. We evaluated the effects of Perilla frutescens leaf extract (PLE) on proliferation and apoptosis inducing in human hepatoma HepG2 cells using a cell proliferation assay, flow cytometry, and cDNA microarrays. Gene expression and apoptosis were also assessed in HepG2 cells treated with a major constituent of PLE, rosmarinic acid (RosA). In the PLE-treated HepG2 cells, antiproliferative activity (105 microg/mL) were observed, flow cytometry revealed significant apoptosis, and microarray data indicated that the expression of a lot apoptosis-related genes were regulated in a time-dependent manner. Compared with PLE, RosA (10 microg/mL; a dose equivalent to 105 microg/mL of PLE) was less effective in increasing the expression of apoptosis-related genes and apoptosis inducing in HepG2 cells. Thus, additional PLE constituents may influence apoptosis in HepG2 cells. The results of our study suggest that the PLE should be further investigated as a promising to treat hepatocellular carcinoma.     

Effect of Sutherlandia frutescens on the Lipid Metabolism in an Insulin Resistant Rat Model and 3T3‐L1 Adipocytes

High fat diet induced insulin resistance correlates with dyslipidaemia and ectopic fat deposits in skeletal muscle and liver. The effects of Sutherlandia frutescens, an antidiabetic medicinal plant, on lipid metabolism were evaluated in an insulin resistant (IR) rat model and in 3 T3‐preadipocytes. Wistar rats received normal diet (ND) or high fat diet (HFD). After the onset of IR in the HFD group, the rats were subdivided into two subgroups, which either continued with HFD or were treated with 50 mg S. frutescens/kg BW/day and HFD (HFD + SF). After 4 weeks, the HFD + SF rats had a significantly lower body weight than the HFD rats (p < 0.05). Blood plasma analysis showed a decrease in insulin, free fatty acids and triglycerides. Related changes in lipid parameters were observed in the liver, skeletal muscle and adipose tissue. To investigate the effects of S. frutescens on adipose tissue, 3 T3‐L1 cells were used as a model. Treatment with S. frutescens led to a decrease in triglyceride accumulation, whilst glucose consumption and lactate production were increased (p < 0.05). These results indicate that S. frutescens directly affects mitochondrial activity and lipid biosynthesis in adipose tissue and provide a mechanism by which S. frutescens can restore insulin sensitivity by modulating fatty acid biosynthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

莫诺苷抑制H2O2诱导的神经细胞凋亡

目的：研究莫诺苷对氧化应急诱导的神经细胞凋亡的影响。方法：培养的人神经母细胞瘤（SH-SY5Y）神经细胞加入莫诺苷（1，10，100μmol&#183;L^-1）预孵育24h，加入H2O2（500μmol&#183;L^-1）作用18h诱导产生氧化损伤，测定对细胞内超氧化物歧化酶（SOD）活性的影响；用Westernblot方法检测caspase-3，caspase-9，Bcl-2和Bax的表达。结果：莫诺苷（10，100μmol&#183;L^-1）抑制氧化损伤，与模型组相比，SOD活性分别增加了14％（P〈0．01）和11％（P〈0．05）；莫诺苷（1，10，100μmol&#183;L^-1）与模型组相比，caspase-3的含量分别减少了31％（P〈0．01），103％（P〈0．001）和95％（P〈0．001），caspase-9的含量分别减少了71％（P〈0．001），132％（P〈0．001）和37％（P〈0．05），Bcl-2分别增加了88％（P〈0．01），121％（P〈0．001）和60％（P〈0．01），对Bax没有影响。结论：莫诺苷可通过抗氧化作用抑制H2O2诱导的神经细胞凋亡，具有神经保护作用。     

Differential signaling involved in Sutherlandia frutescens-induced cell death in MCF-7 and MCF-12A cells

Ethnopharmacological relevance

The scientific study of natural products traditionally used in anticancer preparations has yielded several therapeutically relevant compounds. One of these traditional preparations with potentially beneficial properties is aqueous extracts of Sutherlandia frutescens, a shrub indigenous to the Western Cape region of South Africa. The aims of this study were to evaluate in vitro efficacy of these preparations on the MCF-7 breast adenocarcinoma and MCF-12A non-tumorigenic cell lines in terms of cell proliferation, cell morphology and possible induction of cell death.

Materials and methods

Crystal violet staining was used to evaluate cell proliferation, light-and fluorescence microscopy were used to investigate both intracellular and extracellular morphological features of apoptosis and autophagy (e.g. membrane blebbing, condensed chromatin and intracellular lysosomes), while flow cytometry quantified cell cycle changes and induction of apoptosis through analysis of the flip-flop translocation of phosphatidylserine.

Results

Crystal violet staining showed a time- and dose specific response to aqueous Sutherlandia frutescens extracts, revealing exposure to 1 mg/ml aqueous extract for 48 h to be ideal for comparing the differential effects of Sutherlandia frutescens in the MCF-7 and MCF-12A cell lines. Microscopy showed distinct morphological changes with hallmarks of apoptosis being observed in both cell lines. Flow cytometry revealed a decrease in actively cycling cells in both cell lines, and a 4.36% increase in phosphatidylserine translocation in the MCF-7 cell line, indicative of apoptosis induction, while fluorescence microscopy showed evidence of the induction of autophagy.

Conclusions

Analyses revealed the carcinogenic MCF-7 cell line to be more susceptible to the cytostatic and cytotoxic effects of aqueous extracts of Sutherlandia frutescens when compared to the non-tumorigenic MCF-12A cell line, thus warranting further research into the exact cellular mechanisms involved and the possible synergistic activities of Sutherlandia frutescens ingredients.     

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.     

塞隆骨提取物对成骨样细胞增殖及凋亡作用的研究

目的：研究塞隆骨提取物对体外培养成骨样细胞代谢功能的调节作用及对其凋亡的影响。方法：采用超临界萃取法制备塞隆骨的脂、醇、水提物、水煎提取液及骨胶提取物,以大鼠成骨样细胞ROS 17/2.8为细胞模型,添加塞隆骨各提取物至大鼠成骨细胞培养体系中,MTT法测定成骨样细胞的增殖情况,以流式细胞术检测成骨样细胞凋亡比例。结果：1×10^-2 g·mL^-1塞隆骨脂和水提物对大鼠成骨样细胞ROS 17/2.8有显著的促进增殖作用(P<0.01)；与空白组相比,各塞隆骨提取物使成骨细胞的亚二倍体峰比例明显减少,琼脂糖凝胶电泳结果表明这些提取物能够降低成骨样细胞的凋亡率,推迟凋亡的发生。结论：塞隆骨通过促进成骨样细胞的增殖,降低成骨样细胞的凋亡率和推迟其凋亡的发生,来发挥健骨的作用。     

Effects of pilose antler extracts on doxorubicin-induced beating abnormalities of cultured myocardial cells

Effects of pilose antler extracts and subsequent fractions on normal and doxorubicin (DX)-administered cultured myocardial cells from mouse embryo were investigated. A water extract did not affect the percentage of beating cells at 1–100 μg/mL but suppressed the beating rate at 100 μ/mL. A pretreatment with the water extract significantly restored the percentage of beating cells which was reduced by administration of DX, and showed a tendency to recover the reduced beating rate. A similar pretreatment with an aqueous EtOH-solnble portion (fraction 1) of the water extract significantly recovered both the percentage of beating cells and the beating rate, but further fractionation did not improve the activities. LDH leakage from DX-administered myocardial cells was significantly diminished by the pretreatment with fraction 1, though ATP content in the cells was not appreciably recovered.     

Protective effect of Wen-Pi-Tang against apoptosis of cultured renal epithelial cells

In terms of the rate of DNA fragmentation and electrophoresis patterns, we demonstrated that apoptosis was induced in LLC-PK₁ cells subjected to the Fenton reaction with H₂O₂ and Fe²⁺. In addition, Wen-Pi-Tang, which is known to inhibit the progression of renal failure in both an experimental and a clinical setting, was found to suppress this apoptosis. © 1998 John Wiley & Sons, Ltd.     

Passiflora actinia Hooker extracts and fractions induce catalepsy in mice

Leaves from several Passiflora species are largely employed in the Brazilian folk medicine for its anxiolytic and sedative properties. In behavioral studies, to analyze the tranquillizer action of Passiflora actinia Hooker, it was noteworthy that animals treated with the hydroethanol (HE) and methanol (ME) extracts presented an abnormal postural immobility compared to control animals. That observation led to an investigation of the effects of HE and its fractions on evaluation of catalepsy in mice. The results showed that HE extract, methanol extract, the sequent aqueous crude fractions (AF), and fa, fb and fc chromatographic fractions obtained from Passiflora actinia induced catalepsy in mice. Apparently, the active principles responsible for catalepsy are present in all of the fractions of the extract.     

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.