Propolis from the Stingless Bee Trigona incisa from East Kalimantan,Indonesia, Induces In Vitro Cytotoxicity and Apoptosis in Cancer Cell lines

Background: Previously, stingless bee (Trigona spp.) products from East Kalimantan, Indonesia, weresuccessfully screened for in vitro antiproliferative activity against human cancer derived cell lines. It wasestablished that propolis from T. incisa presented the highest in vitro cytotoxicity against the SW620 coloncancer cell line (6% cell survival in 20 μg/mL). Materials and Methods: Propolis from T. incisa was extractedwith methanol and further partitioned with n-hexane, ethyl acetate and methanol. The in vitro cytotoxicity ofthe extracts was assessed by the MTT assay against human colon (SW620), liver (Hep-G2), gastric (KATO-III),lung (Chago) and breast (BT474) cancer derived cell lines. The active fractions were further enriched by silicagel quick column, absorption and size exclusion chromatography. The purity of each fraction was checked bythin layer chromatography. Cytotoxicity in BT-474 cells induced by cardanol compared to doxorubicin wereevaluated by MTT assay, induction of cell cycle arrest and cell death by flow cytometric analysis of propidiumiodide and annexin-V stained cells. Results: A cardol isomer was found to be the major compound in one activefraction (F45) of T. incisa propolis, with a cytotoxicity against the SW620 (IC50 of 4.51 ± 0.76 μg/mL), KATO-III(IC50 of 6.06 ± 0.39 μg/mL), Hep-G2 (IC50 of 0.71 ± 0.22 μg/mL), Chago I (IC50 of 0.81 ± 0.18 μg/mL) and BT474(IC50 of 4.28 ± 0.14 μg/mL) cell lines. Early apoptosis (programmed cell death) of SW620 cells was induced bythe cardol containing F45 fraction at the IC50 and IC80 concentrations, respectively, within 2-6 h of incubation.In addition, the F45 fraction induced cell cycle arrest at the G1 subphase. Conclusions: Indonesian stingless bee(T. incisa) propolis had moderately potent in vitro anticancer activity on human cancer derived cell lines. Cardolor 5-pentadecyl resorcinol was identified as a major active compound and induced apoptosis in SW620 cells inan early period (≤ 6 h) and cell cycle arrest at the G1 subphase. Thus, cardol is a potential candidate for cancerchemotherapy.     

Biological Activity of Bee Propolis in Health and Diseas

Propolis is a natural product derived from plant resins collected by honeybees. It is used by bees as glue, a ‍general-purpose sealer, and as draught-extruder for beehives. Propolis has been used in folk medicine for centuries. ‍It is known that propolis possesses anti- microbial, antioxidative, anti-ulcer and anti-tumor activities. Therefore, ‍propolis has attracted much attention in recent years as a useful or potential substance used in medicine and cosmetics ‍products. Furthermore, it is now extensively used in foods and beverages with the claim that it can maintain or ‍improve human health. The chemical composition of propolis is quite complicated. More than 300 compounds ‍such as polyphenols, phenolic aldehydes, sequiterpene quinines, coumarins, amino acids, steroids and inorganic ‍compounds have been identified in propolis samples. The contents depend on the collecting location, time and plant ‍source. Consequently, biological activities of propolis gathered from different phytogeographical areas and time ‍periods vary greatly. In this review, the activity of bee propolis will be presented with special emphasis on the ‍antitumor activity.     

Antioxidant and Anti-cancer Cell Proliferation Activity of Propolis Extracts from Two Extraction Methods

Antioxidant activity, total phenolic, total flavonoid compounds and cytotoxicity to cancer cell lines of propolisextracts from two extraction methods were investigated in this study. Propolis was collected from Phayaoprovince and extracted with 70% ethanol using maceration and sonication techniques. The antioxidant activitywas evaluated by DPPH assay. Total phenolic and flavonoid compounds were also determined. Moreover, thecytotoxicity of propolis was evaluated using MTT assay. The percentage propolis yield after extraction usingmaceration (18.1%) was higher than using sonication (15.7%). Nevertheless, antioxidant and flavonoid compoundsof the sonication propolis extract were significant greater than using maceration. Propolis extract from sonicationshowed antioxidant activity by 3.30±0.15 mg gallic acid equivalents/g extract. Total phenolic compound was18.3±3.30 mg gallic acid equivalents/g extract and flavonoid compound was 20.49±0.62 mg quercetin/g extract.Additionally, propolis extracts from two extraction methods demonstrated the inhibitory effect on proliferation ofA549 and HeLa cancer cell lines at 24, 48 and 72 hours in a dose-dependent manner. These results are of interestfor the selection of the most appropriate method for preparation of propolis extracts as potential antioxidantand anticancer agents.     

Antitumoral and Antioxidant Potential of Egyptian Propolis Against the PC3 Prostate Cancer Cell Line

It has been shown previously that nutritional supplements rich in polyphenolic compounds play a significant role in prostate cancer chemoprevention. Propolis is a natural, resinous hive product that has several pharmacological activities including antimicrobial, antioxidant, anti-inflammatory, and antitumoral activities. The aim of this study was to compare the cytotoxic, antioxidant and antitumoral activities of an ethanolic extract of Egyptian propolis (EEP) in vitro with an established chemotherapeutic drug such as doxorubicin (DOX), and the effects of their combination against the PC3 human prostate cancer cell line. Cellular viability and IC50 levels with EEP, DOX and their (v/v) combination were detected by sulphorhodamine-B (SRB) assay after incubation of PC3 cells for 72h with different doses (0, 0.01, 0.1, 1, 10 and 100μg/ml). Two selected doses of IC50 and IC25 were applied to cells for 24h for antitumor evaluation assay of treatment compounds. EEP and its (v/v) combination with DOX showed significant antitumor potential besides high antioxidant properties of superoxide dismutase (SOD), total antioxidant capacity (TAC), catalase (CAT), nitric oxide (NO) and reduced glutathione (GSH) levels when compared with the control untreated cells. DNA fragmentation assay and semi quantitative RT-PCR analyses for p53 and Bax genes showed that EEP activated cellular apoptosis and increased the mRNA expression levels more than other treatment. In conclusion, EEP alone or in combination with DOX at both doses used here showed greater antioxidant, antiproliferative and apoptotic effects against the PC3 cell lines as compared to treatment with DOX alone. Therefore, EEP could be considered as a promising candidate for prostate cancer chemotherapy.     

Ethanolic Extract of Ocimum sanctum Leaves Reduced Invasion and Matrix Metalloproteinase Activity of Head and Neck Cancer Cell Lines

Background: Head and neck squamous cell carcinoma (HNSCC) has a yearly incidence of 600,000 cases worldwide with a low survival rate. Ocimum sanctum L. or Ocimum tenuiflorum L. (Holy basil; Tulsi in Hindi), is a traditional medicine herb that demonstrates numerous effects including anti-oxidant, anti-microbial, and anti-tumor effects. The aim of this study was to evaluate the anti-invasive effect of O. sanctum leaf extract on HNSCC cell lines. Methods: Ethanolic extract of O. sanctum leaf (EEOS) was prepared and the phenolic compounds were identified using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. Genetically matched HNSCC cell lines derived from primary (HN30 and HN4) and metastatic sites (HN31 and HN12) from the same patient were used in this study. The EEOS cytotoxicity to the cell lines was determined using an MTT assay. The invasion and matrix metalloproteinase (MMP)-2 and -9 activity of EEOS-treated cells were tested using a modified Boyden chamber assay and zymography, respectively. Results: We found that EEOS significantly inhibited the invasion and MMP-2 and MMP-9 activity of HN4 and HN12 cells, but not HN30 and HN31 cells. Rosmarinic acid, caffeic acid, and apigenin were detected in EEOS. Moreover, rosmarinic acid was found as the major phenolic compound. Conclusion: EEOS exerted its anti-invasive effect on HNSCC cells by attenuating MMP activity. The active compounds identified in EEOS might be promising as an alternative therapeutic agent for HNSCC.     

Chemo-radioresistance of Small Cell Lung Cancer Cell Lines Derived from Untreated Primary Tumors Obtained by Diagnostic Bronchofiberscopy

New cell lines of small cell lung cancer (SCLC) were established from specimens of untreated primary tumors biopsied by diagnostic bronchofiberscopy. The advantage of this method was ease of obtaining specimens from lung tumors. Establishment of cell lines was successful with 4 of 13 specimens (30%). Clinical responses of the tumors showed considerable variation, but were well correlated with the in vitro sensitivity of the respective cell lines to chemotherapeutic drugs and irradiation. One of the cell lines was resistant to all drugs tested and irradiation, while another was sensitive to all of them. Although the acquired resistance of SCLC is the biggest problem in treatment, the natural resistance to therapy is another significant problem. Either acquired or natural, resistance mechanisms of SCLC may be elucidated by the use of such cell lines derived from untreated tumors. This method and these SCLC cell lines are expected to be useful for the serial study of biologic and genetic changes of untreated and pre-treated tumors, or primary and secondary tumors.     

Anticancer Activity of Solvent Extracts of Hexogonia glabra against Cervical Cancer Cell Lines

Objective: In this study, we aimed to harness some solvent extracts of one wild mushroom Hexagonia glabra and test their anti-cancer activity against cervical human cell lines, namelyHeLa, SiHa, and CaSki. Methods: It includes cell morphological study by microscope, nuclear morphology by DAPI staining under fluorescence microscopy, apoptosis assay by fluorescence technique, anti-proliferation by MTT assay and expression of apoptotic and anti-apoptotic genes by Western blotting and cell cycle analysis was done. Results: The selected cervical cancer cells were treated separately with 150 µg/mL of three extracts, namely of ethanolic (EE), ethyl acetate (EAE), and water extract (WE) and exhibited features like round, shrink and dead. All extracts caused apoptosis in cell lines and EE had the highest effect in this regard. The percentages of apoptotic cells in HeLa, SiHa and CaSki, at the same concentration of EE were 79.23, 75.42, and 76.36% respectively. Cytotoxicity assay showed that all three extracts (50 – 250 μg/mL) were potent for inhibition of cell growth of three cell lines and again EE had the highest effect. The percentages of cell growth inhibition in HeLa, SiHa, and CaSki cells treated with EE at 24 h at 50 µg/mL were 45.79±4.11, 41.66±4.03, and 36.72±2.67, while they were 74.23±7.45, 62.31±5.97, and 54.23±5.04 at 150 µg/mL concentration. At 250 µg/mL concentration, the percentages of cell growth inhibition were 94.25 ±8.11, 90.02 ±8.67, and 85.43±6.21, respectively. The expression of apoptotic gene (Caspase 3, 9) and tumor guard gene (p53), as their proteins in Western blotting increased . However, anti-apoptotic BcL2 gene of all cell lines was decreased following treatment with extracts. In addition, the cell cycle analysis (CaSki cell) showed that treatment (EE) arrested at G2/M check point cell cycle. Conclusion: All extracts of this mushroom were active in arresting growth of three cell lines and EE had the highest effect, indicating that this mushroom can be a valuable source of anticancer agents.     

The Release of Chromogranin A and B Like Activity from Human Lung Cancer Cell Lines: A Potential Marker for a Subset of Small Cell Lung Cancer

Human small cell lung cancer (SCLC) is in vivo and in vitro characterized by a heterogeneous spectrum of neuroendocrine markers. the non-SCLC group is deprived of these markers, or expresses them in low quantities. in this paper we report on the release to the culture medium of neuroendocrine associated proteins, chromogranin A and B like activity (CABLA). the culture medium from three out of five SCLC cell lines and in onehive non-SCLC cell line contained significant levels of CABLA. Normal diploid foreskin fibroblasts and a histiocytic lymphoma cell line were deprived of CABLA production. the presence of CABLA in both SCLC and non-SCLC further stress their common histogenetic origin. the CABLA values were partly unrelated to other neuroendocrine markers. Determinations of CABLA could thus be a potential and valuable marker for a subset of SCLC.     

Pharmacological Activity of Kaempferia parviflora Extract against Human Bile Duct Cancer Cell Lines

crude ethanol extract of Kaemperia parviflora Wall. Ex Baker and a purified compound, 5,7,4-trimethoxyflavone (KP.8.10), were evaluated for pharmacological effects on human cholangiocarcinoma celllines (HuCCA-1 and RMCCA-1). The cells were incubated with various concentrations of extract for varioustime periods and metabolic activity (MTT assay) was assessed for cell viability. The results showed a dosedependenteffect of both crude ethanol extract and the pure compound. CC50s for the crude extract on HuCCA-1 and RMCCA-1 cells were 46.1μg/ml and 62.0μg/ml, respectively. Values for the pure compound could not bedetermined because of solubility problems. Interestingly, K. parviflora ethanol extract and KP.8.10 at lowconcentrations (10-20μg/ml and 2.5-5 μg/ml, respectively) markedly reduced rhHGF-induced invasion byHuCCA-1 and RMCCA-1 cells across matrix-coated transwell plates. Higher concentrations of K. parvifloraethanol extract (60 and 80μg/ml) and KP.8.10 (20 μg /ml) dramatically changed the cellular morphology andcaused death in both cell types. KP.8.10 further exhibited progressive action via caspase-3 mitochondrial enzymeactivation, enhancing cellular toxicity in a time-dose dependent fashion. Therefore, 5,7,4-trimethoxyflavoneappeared to be a bioactive component of K. parviflora extract capable of exerting anti-cancer action. The resultssuggested a benefit of this edible plant in prevention and treatment of cholangiocarcinoma.     

Overexpression of BASP1 Indicates a Poor Prognosis in Head and Neck Squamous Cell Carcinoma

Objective: Brain abundant membrane attached signal protein 1 (BASP1) was originally identified as a membrane and cytoplasmic protein. Recent studies have shown that BASP1 highly expressed in cancer and promoted the proliferation of cancer. However, the role of BASP1 in head and neck squamous cell carcinoma (HNSCC) is largely unknown.  Here, we performed a systematic data analysis to examine whether BASP1 can function as prognostic marker in HNSCC. Methods: In this study, we used Oncomine, and UALCAN, databases to analyze the expression of BASP1 in HNSCC. We used Kaplan-Meier plotter to evaluate the effect of BASP1 on clinical prognosis. In addition, we also analyzed genetic alterations, interaction network, and functional enrichment of BASP1. Results: BASP1 mRNA expression level was remarkably increased in HNSCC than in normal tissues (P=1.624e-12). Moreover, high BASP1 expression was significantly related to poor survival (p=0.00056) in HNSCC patients. In addition, BASP1 gene amplified in 5% of HNSCC patients which contributes to the overexpression of BASP1. Conclusions: These findings suggest that BASP1 was frequently amplified which contributes to the overexpression of BASP1, thereby promoting HNSCC progression. Thus, these results indicate that BASP1 might serve as a biomarker to predict the progression and prognosis of HNSCC patients.     

Establishment of Cell Lines with High and Low Metastatic Potential from A549 Human Lung Adenocarcinoma

This article reports the establishment of variant cell lines with high and low metastatic potential by the dilution plating technique. Two clones with high metastatic potential, 2S Lu-4 and 11S Lu-1 and two clones with low metastatic potential, 8S and 16S were established from A549 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low- metastatic cell lines did not produce lung metastasis after injection into the tail vein of 5-week-old BALB/c nude mice. The primary tumors produced by the two high-metastatic cells grew fast and showed enhanced angiogenesis. The high-metastatic cells were small and flat-shaped, while the low- metastatic cells were large and flat-shaped. When the four variant cell lines and original A549 cells were embedded in collagen gels, the colonies of 2S Lu-4, 11S Lu-1 and A549 grew actively, whereas almost of all the colonies of 8S and 16S did not survive after 35 days in culture. These four cloned cell lines originated from heterogeneous populations of the parental A549 cells should be an excellent tool for studying the process of metastasis of lung cancer.     

Cytotoxic and Apoptotic Effects of Extracts of Artemisia ciniformis Krasch. & Popov ex Poljakov on K562 and HL-60 Cell Lines

Artemisia, as one of the largest genera in the tribe Anthemideae of the Asteraceae comprises an important part of Iranian flora. While cytotoxic and apoptotic properties have already been reported for some species of the genus there is not any report on cytotoxic effects of A. ciniformis. Petroleum ether (40-60), dichloromethane, ethyl acetate, ethanol and ethanol-water (50:50) extracts of the aerial parts of A. cinformis were subjected to cytotoxic and apoptotic evaluations on two cancer human cell lines (K562 and HL-60) and on J774 normal cells.Among multiple extracts evaluated for cytotoxicity, dichloromethane (CH2Cl2) and petroleum ether (PE) extracts were shown to possess the highest anti-proliferative effects on HL-60 and K562 cells with IC50 values of 31.3 and 25.5 μg/ml respectively. Apoptosis induction verified by sub-G1 peaks was seen in flow cytometry histograms. Increase in the amount of Bax protein, formation of DNA fragments, and cleavage of PARP to 24 and 89kDa sub units all confirmed induction of apoptosis by A. cinformis extracts. Taken together according to the result of the present study some extracts of A. cinformis could be considered as sources for natural cytotoxic compounds and further mechanistic and phytochemical studies are recommended to fully understand the underlying mechanisms of cnacer cell death as well as identification of responsible phytochemicals.     

Prognostic Significance of Epidermal Growth Factor Receptor Phosphorylation and Mutation in Head and Neck Squamous Cell Carcinoma

The molecular status of the epidermal growth factor receptor (EGFR) has not been as well studied in head and neck squamous cell carcinoma (HNSCC) as in lung cancer. We examined the frequencies of EGFR mutations as well as the expression/phosphorylation status of the EGFR protein in HNSCC patients. Moreover, we tried to elucidate associations between EGFR molecular status and patient characteristics and disease‐free survival. In this prospective cohort study, clinical data and samples were obtained from 82 consecutive patients who had not been treated with EGFR molecular targeting therapy. Full‐length EGFR was sequenced, and expression and phosphorylation of the EGFR protein were measured by Western blotting. Four novel mutations (E709K, V765G, Ins770G, and G1022S) and one mutation well‐known in lung cancer (L858R) were identified in six HNSCC samples (7%), but we could not find any mutations in the extracellular domain of EGFR, such as EGFRvIII, in this study. E709K and Ins770G as well as L858R appear to be functional mutations based on the use of Ba/F3 cells. In terms of patient characteristics, the number of metastatic lymph nodes and node stage were associated with phosphorylation of EGFR. No patients with EGFR mutations relapsed during the study period. Excluding mutated cases, patients whose tumor samples showed phosphorylated EGFR relapsed significantly earlier than those without phosphorylated EGFR. This finding was still significant after adjusting for mutation and overexpression of EGFR protein using the Cox proportional hazard model. In conclusion, phosphorylated EGFR without mutations may be a marker of poor prognosis in patients with HNSCC.     

Analysis of T Cell Receptor Variability in Fresh Tumor-infiltrating Lymphocytes from Human Head and Neck Cancer

In this study, we analyzed T cell receptor (TCR) gene rearrangements in tumor-infiltrating lymphocytes (TIL) freshly obtained from 15 patients with head and neck cancer using the reversely transcribed polymerase chain reaction (RT-PCR) method. These TILs showed preferential expression of Vα10, Vα8 and Vα1, detected in 13 (87%), 11 (73%), and 9 cases (60%), respectively. The TCRVβ gene revealed diversity without preferential usage. The head and neck region is exposed to bacteria and viruses, so it is possible that the tumor site can become infected and accumulate T cells involved in infection and inflammation. Therefore, we also investigated TCR gene usage in T cells infiltrating in chronic sinusitis mucosa to address the question of whether the Vα1, Vα8, and Vα10 subfamilies are characteristic in TIL from squamous cell carcinoma of head and neck. TCR Vα10 gene usage was also the most common in Vα segment in T cells infiltrating the sinus mucosa, but Vα and Vα8 were not detected in the T cells in sinusitis. These results indicate that the Vα10 subfamily, the preferred T cell population in both TIL and T cells in inflammatory disease, might he involved mainly in inflammation or infection. On the other hand, Vα1 and Vα8 appear to be relatively specific populations for antitumor immunity in head and neck cancer.     

Evaluation of In Vitro Cytotoxic Property Against Cholangiocarcinoma Cell Line and GC/MS Analysis from Leaf of Erythrophleum succirubrum Gagnep

Objective: Plants are valuable sources of new pharmaceuticals. Secondary metabolites of the genus Erythrophleum exhibit cytotoxicity and may have therapeutic value. The cytotoxic activity of ethanolic leaf extract of Erythrophleum succirubrum Gagnep. against a human cholangiocarcinoma cell line was assessed. Methods: Crude extract of E. succirubrum was prepared by ethanol extraction. The ethanolic leaf extract of E. succirubrum was evaluated for cytotoxicity against the human cholangiocarcinoma cell line KKU-M213 using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays. The chemical composition of E. succirubrum leaf extract was analyzed using GC/MS. Result: The ethanolic leaf extract of E. succirubrum reduced the viability of KKU-M213 cells in a dose- and time-dependent manner. It showed high cytotoxicity, with IC50 values of 65.22 ± 1.18 µg/mL and 1.19 ± 1.38 µg/mL at exposure times of 24 and 96 h, respectively. GC/MS analysis of the ethanolic leaf extract of E. succirubrum identified 22 components. The main constituents identified were Cyclohexanone, 2-[2-nitro-1-(2-naphthyl)ethyl]-(14.79%) followed by allomycin (14.65%), mome inositol (14.30%), campesterol (11.80%) and ethyl linolenate (10.83%), respectively. Conclusion: Five major groups of compounds were found, with lipids dominating, followed by carbohydrates, benzenoids, phenylpropanoids, polyketides and organoheterocyclic compounds. Many of the bioactive components discovered in the ethanolic leaf extract of E. succirubrum might be responsible for its cytotoxic properties.     

Metastatic Squamous Cell Carcinoma of an Unknown Primary Tumor Localized to the Neck

68 patients with metastatic squamous-cell carcinoma (SCC) of an unknown primary tumor localized to the neck were treated between 1981 and 1990. There were 11 patients treated with radiotherapy alone, 24 patients treated with surgery and radiotherapy and 33 patients treated with radiotherapy and chemotherapy. Male to female ratio was 1.9 : 1 and the median age was 55 years (range, 33 to 71 years). 41 (61%) patients had N3 disease, 18 (26%) patients had N2 disease and 9 (13%) patients had N1 disease. The majority of N3 patients were treated with radiotherapy + chemotherapy (n=17) and surgery + radiotherapy (n=17). The complete response (CR) to radiotherapy + chemotherapy was 73% with 19 patients having no evidence of disease currently. The median survival time (MST of this group was 34+ months. Of the 35 patients who had surgery and/or radiotherapy, 7 (20%) currently have no evident disease. The MST of these two groups (combined) was 22 months. Patients with N3 disease who received radiotherapy + chemotherapy had a higher CR rate and longer MST when compared with those without chemotherapy.     

Cytotoxic and Apoptotic-inducing Effects of Purple Rice Extracts and Chemotherapeutic Drugs on Human Cancer Cell Lines

Pigmented rice is mainly black, red, and dark purple, and contains a variety of flavones, tannin, polyphenols,sterols, tocopherols, γ-oryzanols, amino acids, and essential oils. The present study evaluated the cytotoxic effectsof purple rice extracts (PREs) combined with chemotherapeutic drugs on human cancer cells and mechanismsof cell death. Methanolic (MeOH) and dichloromethane (DCM) extracts of three cultivars of purple rice inThailand: Doisaket (DSK), Nan and Payao (PYO), were tested and compared with white rice (KK6). Cytotoxicitywas determined by 3-(4, 5-dimethyl)-2, 5-diphenyltetrazolium bromide (MTT) assay in human hepatocellularcarcinoma HepG2, prostate cancer LNCaP and murine normal fibroblast NIH3T3 cells. MeOH-PYO-PRE wasthe most cytotoxic and inhibited HepG2 cell growth more than that of LNCaP cells but was not toxic to NIH3T3cells. When PREs were combined with paclitaxel or vinblastine, they showed additive cytotoxic effects on HepG2and LNCaP cells, except for MeOH-PYO-PRE which showed synergistic effects on HepG2 cells when combinedwith vinblastine. MeOH-PYO-PRE plus vinblastine induced HepG2 cell apoptosis with loss of mitochondrialtransmembrane potential (MTP) but no ROS production. MeOH-PYO-PRE-treated HepG2 cells underwentapoptosis via caspase-9 and-3 activation. The level of γ-oryzanol was highest in DCM-PYO-PRE (44.17 mg/g)whereas anthocyanin content was high in MeOH-PYO-PRE (5.80 mg/g). In conclusion, methanolic Payaopurple rice extract was mostly toxic to human HepG2 cells and synergistically enhanced the cytotoxicity ofvinblastine. Human HepG2 cell apoptosis induced by MeOH-PYO-PRE and vinblastine was mediated througha mitochondrial pathway.