Castasterone, a Plant Steroid Hormone, Affects Human Small-Cell Lung Cancer Cells and Reverses Multi-Drug Resistance.

Small-cell lung cancer (SCLC) has a dismal prognosis, in part because of the development of multi-drug resistance. Castasterone (CAS) is the metabolic precursor of the plant steroid hormone epibrassinolide (EB). In some plants, EB accounts for the total hormone activity, whereas in other plants, CAS is the active form. The effects of CAS, a BR present in most plants, on animal cells in general and cancer cells in particular have not been described. Here, we report the effects of CAS on drug-sensitive (H69) and drug-resistant (VPA17) SCLC cells. CAS was equally cytotoxic to both cell lines (IC50 = 1 µM), indicating a lack of cross-resistance. Pre-incubation of VPA17 cells with CAS for 96 h reversed drug resistance to etoposide and doxorubicin. Synergism between CAS and EB, as well as with chemotherapy drugs, was investigated by exposure of VPA17 cells to 1:1 ratios of CAS and the other drugs at the respective IC50 values, with dilutions at 0.25 to 2.0 × IC50 and determination of the combination index (CI). CAS and EB were additive, indicating that the two drugs act on the same pathway, whereas CAS-etoposide (CI = 0.77) and CAS-doxorubicin were synergistic, indicating that CAS and the two chemotherapeutic drugs act on different pathways. Apoptosis in SCLC cells was measured by immuno-detection of single-strand DNA breaks. Following 96 h incubation of SCLC H69 cells in CAS, the level of DNA breaks was similar to measurements made after incubation in EB and etoposide, indicating that CAS is pro-apoptotic. Incubation of SCLC cells in CAS led to a time-dependent reduction (by 80%) in the transcriptional activator ß-catenin. These data indicate that CAS may act via Wnt signaling. Taken together, our study reveals that CAS is pharmacologically active in both drug-sensitive and drug-resistant SCLC cells.

Mechanism-Driven and Clinically Focused Development of Botanical Foods as Multitarget Anticancer Medicine: Collective Perspectives and Insights from Preclinical Studies, IND Applications and Early-Phase Clinical Trials.

Cancer progression and mortality remain challenging because of current obstacles and limitations in cancer treatment. Continuous efforts are being made to explore complementary and alternative approaches to alleviate the suffering of cancer patients. Epidemiological and nutritional studies have indicated that consuming botanical foods is linked to a lower risk of cancer incidence and/or improved cancer prognosis after diagnosis. From these observations, a variety of preclinical and clinical studies have been carried out to evaluate the potential of botanical food products as anticancer medicines. Unfortunately, many investigations have been poorly designed, and encouraging preclinical results have not been translated into clinical success. Botanical products contain a wide variety of chemicals, making them more difficult to study than traditional drugs. In this review, with the consideration of the regulatory framework of the USFDA, we share our collective experiences and lessons learned from 20 years of defining anticancer foods, focusing on the critical aspects of preclinical studies that are required for an IND application, as well as the checkpoints needed for early-phase clinical trials. We recommend a developmental pipeline that is based on mechanisms and clinical considerations.

Exploring the Biological Activity and Mechanism of Xenoestrogens and Phytoestrogens in Cancers: Emerging Methods and Concepts.

Xenoestrogens and phytoestrogens are referred to as "foreign estrogens" that are produced outside of the human body and have been shown to exert estrogen-like activity. Xenoestrogens are synthetic industrial chemicals, whereas phytoestrogens are chemicals present in the plant. Considering that these environmental estrogen mimics potentially promote hormone-related cancers, an understanding of how they interact with estrogenic pathways in human cells is crucial to resolve their possible impacts in cancer. Here, we conducted an extensive literature evaluation on the origins of these chemicals, emerging research techniques, updated molecular mechanisms, and ongoing clinical studies of estrogen mimics in human cancers. In this review, we describe new applications of patient-derived xenograft (PDX) models and single-cell RNA sequencing (scRNA-seq) techniques in shaping the current knowledge. At the molecular and cellular levels, we provide comprehensive and up-to-date insights into the mechanism of xenoestrogens and phytoestrogens in modulating the hallmarks of cancer. At the systemic level, we bring the emerging concept of window of susceptibility (WOS) into focus. WOS is the critical timing during the female lifespan that includes the prenatal, pubertal, pregnancy, and menopausal transition periods, during which the mammary glands are more sensitive to environmental exposures. Lastly, we reviewed 18 clinical trials on the application of phytoestrogens in the prevention or treatment of different cancers, conducted from 2002 to the present, and provide evidence-based perspectives on the clinical applications of phytoestrogens in cancers. Further research with carefully thought-through concepts and advanced methods on environmental estrogens will help to improve understanding for the identification of environmental influences, as well as provide novel mechanisms to guide the development of prevention and therapeutic approaches for human cancers.

The effect of brassinolide, a plant steroid hormone, on drug resistant small-cell lung carcinoma cells.

Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Epibrassinolide (EB) is a steroid hormone in plants, with many physiological effects. It acts via a membrane receptor and GSK3 pathway, resulting in stabilization of a transcription factor. The parallels to the Wnt signaling pathway, which is activated in SCLC and results in increased ß-catenin, prompted investigations of the effects of EB on drug-resistant (VPA17) and drug-sensitive (H69) SCLC cells. EB was cytotoxic to both cell lines (IC50 = 2 µM), indicating a lack of cross-resistance in the VPA17 cell line. EB was pro-apoptotic after 24 h as measured by ELISA of BUdR-labeled DNA fragments and caspase-3 specific activity (2.5 enzyme units/mg protein vs. 0.01 units/mg protein for untreated controls). Matrigel assays showed that EB reduced the SCLC cell invasion phenotype by 80%. Pre-incubation of VPA17 cells in 1 µM EB for 96 h reversed resistance to etoposide (IC50 = 6.0 µM, reduced to 1.8 µM with EB) and doxorubicin (IC50 = 0.37 µM, reduced to 0.09 µM). Synergism between EB and chemotherapy drugs was investigated by exposure of VPA17 cells to 1:1 ratios at the respective IC50 values, with serial dilutions at 0.25 to 2.0 × IC50 and determination of the combination index (CI). EB and etoposide showed synergism (CI = 0.80 at ED50); EB and doxorubicin also showed synergism (CI = 0.65 at ED50). Incubation of SCLC cells in EB led to a time- and dose dependent reduction of ß-catenin (maximum 80% reduction). Gene expression analyses of SCLC cells showed EB incubation resulted in significant reduction in expression of ß-catenin-dependent genes that are anti-apoptotic (e.g., c-Jun, survivin), cell division-related (e.g., CCND1 cyclin, sox9), and metastasis-related (e.g., MMP7, uPAR). WIKI4, a known inhibitor of Wnt signaling, was cytotoxic to SCLC cells (IC50 = 0.02 µM). Synergism between EB and WIKI4 was determined by the CI method and showed antagonism (CI = 1.09 at ED50), suggesting that EB and WIKI4 act on the same pathway. Taken together, these data indicate that EB, a natural product with widespread occurrence in plants, is pharmacologically active in both drug-sensitive and drug-resistant SCLC cells and acts through the Wnt signaling pathway.

Silibinin reverses drug resistance in human small-cell lung carcinoma cells.

Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Silibinin is a flavonolignan extracted from milk thistle (Silybum marianum), extracts of which are used in traditional medicine. We tested the effects of silibinin on drug-sensitive (H69) and multi-drug resistant (VPA17) SCLC cells. VPA17 cells did not show resistance to silibinin (IC50 = 60 µM for H69 and VPA17). Flow cytometry analysis after incubation in 30 µM silibinin showed no changes in cell cycle phases in VPA17 or H69 cells compared with untreated cells. Silibinin (30 µM) incubation was pro-apoptotic in VPA17 cells after > 3 days, as measured by ELISA of BUdR labeled DNA fragments. Apoptosis was also indicated by an increase in caspase-3 specific activity and decrease in survivin in VPA17 MDR cells. VPA17 cells had increased Pgp-mediated efflux of calcein acetoxymethyl ester (calcein AM); however, this was inhibited in cells pre-incubated in silibinin for 5 days. Pre-incubation of VPA17 cells in 30 µM silibinin for 5 days also reversed resistance to etoposide (IC50 = 5.50 to 0.65 µM) and doxorubicin (IC50 = 0.625 to 0.035 µM). The possible synergistic relationship between silibinin and chemotherapy drugs was determined by exposure of VPA17 cells to 1:1 ratios of their respective IC50 values, with serial dilutions at 0.25 to 2.0 × IC50 and calculation of the combination index (CI). Silibinin and etoposide showed synergism (CI = 0.46 at ED50), as did silibinin and doxorubicin (CI = 0.24 at ED50). These data indicate that in SCLC, silibinin is pro-apoptotic, reverses MDR and acts synergistically with chemotherapy drugs. Silibinin, a non-toxic natural product may be useful in the treatment of drug-resistant SCLC.

Effect of Ganoderma on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells.

Multidrug resistance is a major problem in small-cell lung cancer (SCLC). Ganoderma lucidum is a widely used herb in traditional Chinese medicine. We tested the effects of Ganoderma on drug-sensitive (H69) and multi-drug resistant (VPA) human SCLC cells. Both cells showed equal cytotoxicity when incubated with extracts of mycelia of 9 species of Ganoderma, including G. lucidum. Cells treated with the IC(50) of cytotoxic Ganoderma and analyzed by flow cytometry-PI staining showed increases in S phase. When compared untreated controls or SCLC cells treated with extracts of non-cytotoxic Ganoderma species, cells treated with extracts of cytotoxic Ganoderma species responded with an induction of apoptosis similar to cells treated with the chemotherapeutic drugs etoposide and doxorubicin. This was shown by four criteria: increased DNA fragmentation within cells as measured by ELISA; increased TUNEL staining for DNA breaks; increased specific activities of caspases 3 and 9, but not caspase 8 by colorimetric assays, indicating the endogenous pathway; and similar patterns changes in the expressions of 9 genes involved in the cell cycle/apoptosis, as measured by RT-PCR and capillary electrophoresis. Pre-incubation of drug-resistant SCLC cells with cytotoxic Ganoderma reduced the IC(50) for etoposide (3.4-0.21 microM) and doxorubicin (0.19-0.04 microM). These results show that extracts of several species of Ganoderma are cytotoxic to both drug-sensitive and drug-resistant SCLC cells, are pro-apoptotic, induce gene expression patterns that are similar to SCLC cells treated with chemotherapeutic drugs, and can reverse resistance to chemotherapeutic drugs.

The green tea polyphenol, epigallocatechin-3-gallate inhibits telomerase and induces apoptosis in drug-resistant lung cancer cells.

Epidemiological studies on humans and investigations in animal models suggest that consumption of green tea has anti-cancer effects. Small-cell lung carcinoma (SCLC) has a poor prognosis, particularly due to the development of drug resistance. We investigated the effects of the green tea polyphenol, epigallocatechin-3-gallate (EGCG) on human SCLC cells. EGCG had similar effects (IC(50) of approximately 70 microM) on drug-sensitive (H69) and drug-resistant (H69VP) SCLC cells, indicating that it is not part of the drug resistance phenotype expressed in these cells. In both cell lines, incubation in EGCG at 1 x IC(50) for 24h resulted in 50-60% reduced telomerase activity as measured by a PCR-based assay for telomeric repeats. Colorimetric assays of cells treated for 36 h with EGCG demonstrated a reduction in activities of caspases 3 (50%) and 9 (70%) but not caspase 8, indicating initiation of apoptosis. DNA fragmentation as measured by ELISA occurred within cells treated with EGCG and this was confirmed by TUNEL staining. Flow cytometric analysis of SCLC cells incubated for 36 h in EGCG indicated a cell-cycle block in S phase. These data indicate the potential use of EGCG, and possibly green tea, in treating SCLC.

Contaminants of PC-SPES are not responsible for cytotoxicity in human small-cell lung carcinoma cells.

PC-SPES is a mixture of herbs used in the treatment of prostate cancer. Batches of this product were found to contain traces of synthetic drugs, and the product was removed from the market. On the basis of a correlation between contaminant levels and cytotoxicity in prostate carcinoma cell lines, Sovak et al. [M. Sovak, A. Seligson, M. Konas, M. Hajduch, M. Dolezal, M. Machala, R. Nagourney, Herbal composition PC-SPES for management of prostate cancer: identification of active principles, J. Natl Cancer Inst. 94 (2002) 1275-1281] concluded that the contaminants were responsible for cytotoxicity of this preparation. Previously, we showed that extracts of PC-SPES are cytotoxic and pro-apoptotic in both drug-sensitive (H69) and drug resistant (H69V) human small-cell lung carcinoma (SCLC) cell lines. Here, we investigated whether the contaminants might be responsible for these effects. In contrast to the data reported for prostate carcinoma cells, extracts of batches of PC-SPES from the year 1998 (reportedly contaminated) and 2001 (much less contaminated) were equally cytotoxic in both SCLC cell lines. Tests of individual contaminants gave IC50 values far in excess of the amounts reported to be present in the IC50 level for the PC-SPES extracts: diethlystilbesterol: actual IC50 in H69 cells, >1000 microM; concentration present in herbal extract at IC50, 0.05-0.2 microM; indomethacin: actual IC50 in H69 cells, 800 microM; concentration in herbal extract, 1.5-20 microM; warfarin: actual IC50 in H69 cells, 950 microM; concentration in herbal extract, 0.57-0.93 microM. Adding the calculated maximum concentration of the contaminants, singly or in combination, to extracts of the less contaminated batch (2001) of PC-SPES did not alter the cytotoxicity of the extract in H69 or H69V cells. At the contaminated concentrations, as well as 5 x those concentrations, none of the contaminants was pro-apoptotic, as indicated by a DNA fragmentation kinetics assay. However, extracts of both early and late batches of PC-SPES were pro-apoptotic in SCLC cells. We conclude that the traces of pharmaceuticals found in PC-SPES were not responsible for its cytotoxic and pro-apoptotic activities of this herbal mixture on SCLC cells.

Pancreatic cancer in vitro toxicity mediated by Chinese herbs SPES and PC-SPES: implications for monotherapy and combination treatment.

One of the greatest challenges in the treatment of pancreatic cancer remains its inherent lack of beneficial response to cytotoxic chemotherapy. Chinese herbal extracts have been widely used for the treatment of various cancers, but objective information on their efficacy in pancreatic cancer is lacking. Eight human pancreatic cancer cell lines (MIA, Panc-1, BxPC, ASPC, HS-766T, CaPan-2, CFPAC, and HTB-147) were studied for in vitro susceptibility to ethanol extracts of SPES and PC-SPES, two quality-controlled, dried, encapsulated supplements of 15 and eight Chinese herbs, respectively. Resulting toxicities, alone and in combination with doxorubicin or gemcitabine, were analyzed by [(3)H]thymidine incorporation or sulforhodamine B staining, colony formation, and TUNEL flow cytometry assays. Combination toxicity mechanisms were calculated by the combination index method of Chou and Talalay. In all cell lines, there was dose-dependent inhibition of proliferation. By [(3)H]thymidine incorporation assay, 50% growth inhibition after 48 h continuous exposure (IC(50)) occurred at concentrations of 0.2-0.8 microl/ml SPES and 0.4-1.3 microl/ml PC-SPES. Growth inhibition was accompanied by a significant enhancement of the TUNEL-positive apoptotic fraction of all cell lines after treatment with either extract. After treatment with PC-SPES, the cell lines consistently displayed a G2 cell cycle block; SPES induced an increase in S phase, with a smaller impact on G2. When added at a concentration of 0.2 microl/ml (approximately IC(20)), both extracts enhanced Panc-1 cell killing mediated by doxorubicin, with an average decrease in the corresponding IC(50) of 33% (range 11-62%). Combination effects with either extract appeared to be antagonistic in the case of gemcitabine and additive to mildly synergistic in the case of doxorubicin. Both SPES and PC-SPES exhibited significant toxicity in pancreatic cancer cells, mediated via induction of apoptosis. Both mixtures should be evaluated for their in vivo and clinical therapeutic utility as monotherapy agents against pancreatic cancer. SPES could possibly be combined with cell cycle-independent cytotoxic drugs. Due to a consistent G2 blocking pattern, PC-SPES may prove useful as a radiation sensitizer.

Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells.

Multiple drug resistance is a significant problem in small-cell lung cancer (SCLC). Artemisinin (ART) is a natural product used to treat drug-resistant malaria. The drug is effective because the Fe2+ present in infected erythrocytes acts non-enzymatically to convert ART to toxic products. We tested the effects of ART on drug-sensitive (H69) and multi-drug-resistant (H69VP) SCLC cells, pretreated with transferrin (TF) to increase the intracellular Fe2+ level. Antibody staining followed by flow cytometry analysis showed twice the level of TF receptors on the H69VP as compared to the H69 cells. Low doses of ART were cytotoxic to SCLC cells. The cytotoxicity of ART for H69VP cells (IC50=24 nM) was ten-fold lower than for H69 cells (IC50=2.3 nM), indicating that ART is part of the drug resistance phenotype. Pretreatment of H69 cells with 220-880 nM TF did not alter the IC50 for ART. However, in the ART-resistant H69VP cells, pretreatment with TF lowered the ART IC50 to near drug-sensitive levels (IC50=5.4 nM after 4 h pretreatment with 880 nM TF). Desferrioxamine (5 microM) inhibited the effect of TF on the IC50 for ART in drug-resistant cells but did not have an effect on ART cytotoxicity in drug-sensitive cells. DNA fragmentation as measured by ELISA occurred within ART-treated cells, with kinetics indicating apoptosis rather than necrosis. This was confirmed by TUNEL staining. These data indicate the potential use of ART and TF in drug-resistant SCLC.

Effects of four Chinese herbal extracts on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells.

PURPOSE: We examined the pharmacology, cell biology and molecular biology of small-cell lung carcinoma cells treated with four extracts of Chinese herbal medicines. Many cancer patients take these medicines, but their effects at the cellular level are largely unknown. We were especially interested in the effects on drug-resistant cells, as resistance is a significant clinical problem in lung cancer. METHODS: Drug-sensitive (H69), multidrug-resistant (H69VP) and normal lung epithelial cells (BEAS-2) were exposed to extracts from two plants used in Chinese herbal medicine for lung cancer: Glycorrhiza glabra (GLYC) and Olenandria diffusa (OLEN), and to extracts of two commercially available combinations of Chinese herbal medicines, SPES (15 herbs) and PC-SPES (8 herbs). Cytotoxicity was measured in terms of cell growth inhibition (IC(50)). The kinetics of DNA fragmentation after exposure to the herbal extracts was measured by BudR labeling followed by ELISA. Apoptosis was measured by the TUNEL assay followed by flow cytometry. Expression of apoptosis- and cell cycle-related genes was measured by reverse transcription of mRNA followed by filter hybridization to arrays of probes and detection by chemiluminescence. RESULTS: In each case, the four herbal extracts were equally cytotoxic to H69 and H69VP and less cytotoxic to BEAS-2. All four extracts induced DNA fragmentation in the lung carcinoma cells. The kinetics showed DNA fragments released to the medium (an indication of necrosis) in GLYC-exposed cultures, but inside the cells (an indication of apoptosis) in OLEN-, SPES- and PC-SPES-exposed cultures. TUNEL analysis confirmed that exposure to the latter three extracts, but not to GLYC, led to apoptosis. Compared to untreated and GLYC-treated cells, H69 and H69VP cells treated with OLEN, SPES and PC-SPES showed elevated expression of a number of genes involved in the apoptotic cascade, similar to cells treated with etoposide and vincristine. CONCLUSION: The Chinese herbal medicine extracts OLEN, SPES and PC-SPES are cytotoxic to both drug-resistant and drug-sensitive lung cancer cells, show some tumor cell specificity compared to their effect on normal cells, and are proapoptotic as measured by DNA breaks and gene expression. The reaction of the tumor cells to these extracts was similar to their reaction to conventional chemotherapeutic drugs.

Liposomal daunorubicin overcomes drug resistance in human breast, ovarian and lung carcinoma cells.

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC50 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC50 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC50 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC50 of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC50 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC50 237 nM) and H69VP small-cell lung carcinoma cells (IC50 27 nM). Empty liposomes did not affect the IC50 for free DR in the three resistant cell lines, nor did empty liposomes affect the IC50 for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.