Effects Of Four Chinese Herbal Extracts On Drug-sensitive And Multidrug-resistant Small-cell Lung Carcinoma Cells

David Sadava Julie Ahn Mei Zhan Mei-Lin Pang Jane Ding Susan E. Kane

Abstract 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), multi-drug-resistant (H69VP), and normal lung epithelial cells (BEAS-2) were exposed to extracts from two plants used in Chinese herbal medicine for lung cancer: Glycyrrhiza 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 (IC50). 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, showing 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 ex- tracts was similar to their reaction to conventional chemotherapeutic drugs.

Cistanche, a precious Chinese herbal medicine, has the effect of enhancing immunity and lung cancer. Cistanche has a long history of medicinal use. It was first recorded in "Shen Nong's Materia Medica". Shennong tasted all kinds of herbs. The efficacy and role of Cistanche are significant in invigorating the kidney and nourishing essence, relieving fatigue, moistening the intestines and defecation, nourishing yang and nourishing yin, etc., so it was recorded in the book. During the post-Tang Dynasty, Cistanche was listed as one of the nine Chinese fairy types of grass. Modern research on it has been carried out for more than 60 years. , Anti-aging, helps to enhance memory and immunity, etc.

Keywords: Lung cancer, Drug resistance, Chinese herbal medicines, cistanche

Contact: joanna.jia@wecistanche.com

Introduction

Small-cell lung cancer (SCLC) accounts for about 20% of all lung cancers and is aggressive, with a 5-year survival rate at diagnosis of less than 10% [19]. Patients commonly present with disseminated disease, and so treatment is by chemotherapy, with combinations commonly involving cisplatin, etoposide, doxorubicin, 5-fluorouracil, and taxol [12]. Unfortunately, this treatment eventually becomes ineffective because most SCLC tumors develop multidrug resistance [18]. Since there are many resistance mechanisms [15], overcoming resistance is a major clinical challenge.

Faced with palliative care, many cancer patients use alternative medicines, including herbal therapies [6, 23]. Among these therapies, traditional Chinese medicine is probably the best established and codified, dating back several thousand years. The theoretical basis of this medical system concerns the opposing forces of yin and yang, generative and destructive cycles, and a life force, or qi [2]. Specific herbal extracts, and combinations, have been devised to treat specific diseases including cancers [11, 27]. Although there is some empirical evidence that the herbs are effective in treating cancer, their mechanisms of action at the cellular level are largely unknown. This takes on added importance if patients are combining their use with conventional chemotherapy. Also, the effects of herbal extracts on drug resistance have not been reported.

We investigated the effects of four Chinese herbal medicines on three cell lines: SCLC, multidrug-resistant SCLC, and normal lung epithelium. Two of the four extracts were from single plants often prescribed for lung cancer, Glycyrrhiza glabra (GLYC) and Olenandria dif- fusa (OLEN). The other two were from commercially available combinations of herbs called SPES and PC- SPES. OLEN (Chinese name Bai Hua she cao) has antitumor, antimutagenic, and immunostimulating activities [1, 25, 29]. GLYC (Chinese name gan car) is antiinflammatory, antitumorigenic, and antimutagenic [7, 11]. SPES, developed by BotanicLabs (Brea, Calif.), is in the form of capsules containing lyophilized extracts of 15 Chinese herbs and has been shown to decrease pain in patients with advanced cancers [14]. Among the herbs in SPES are the immune stimulant Cistanche deserticola, the antitumor agent Rabdosia rubescens, and the anti-inflammatory agent Zanthoxylum iridium [1, 11]. PC- SPES, also prepared by BotanicLabs, contains eight herbs, including the antiandrogenic Serenoa repens that inhibits prostatic hyperplasia [21], the protein kinase C inhibitor Scutellaria baicalensis [13], the antitumor agent Panax ginseng [30], and Glycyrrhiza glabra (see above). While rigorous evidence for clinical antitumor efficacy of GLYC, OLEN, and SPES is lacking, there is some evidence for the efficacy of PC-SPES. In both prostate cancer cells and animal models, PC-SPES is proapoptotic and cytotoxic [8, 9, 10, 28]. Clinical trials have indicated that it is effective in lowering levels of prostate- specific antigen and in stopping tumor growth in both androgen-dependent and androgen-independent prostate cancer [4, 5, 17, 20, 26].

Although cancer patients doubtless use herbal ex- tracts, there is scant preclinical or clinical data on the effects of these extracts on SCLC. Our goal was to begin this analysis with an examination of the pharmacology (cytotoxicity), cell biology (method of cell death), and molecular biology (gene expression) in drug-sensitive and drug-resistant SCLC cells.

Materials and methods

Extracts and chemicals

GLYC and OLEN were obtained as dried plants from Jen-On Medical Group (Monterey Park, Calif.). For the preparation of an extract, 0.5 g was ground to a fine powder with a mortar and pestle and suspended in 30 ml distilled water. In Chinese medicine, the plant extracts are consumed as teas after heating. Therefore, the suspension was incubated with shaking at 70°C for 18 h. Following centrifugation at 1500 g for 10 min, the supernatant was sterilized by filtering through a 0.45-lm filter using a syringe. The resulting extract was adjusted with distilled water to 17 mg/ml based on the original plant material. The extracts were stored at 4°C for up to 1 week until use.

SPES and PC-SPES were obtained from BotanicLabs (Brea, Calif.) as capsules. The extraction method used has been employed in previous studies of these preparations [8, 9, 10]. The contents of one capsule (320 mg) were dissolved in 1 ml 95% ethanol and the suspension was incubated with shaking at 37°C for 1 h. After centrifugation at 3000 g for 10 min, the supernatant was filter-sterilized as above. The final concentration was 300 mg/ml based on the original plant material. The extracts were stored at –20°C for up to 1 week until use.

Cell cultures and cytotoxicity measurement

NCI-H69 SCLC cells were grown at 37°C in an atmosphere containing 5% CO2 as a suspension in AIM-V serum-free medium (Life Technologies, Grand Island, N.Y.). A multidrug-resistant cell line (H69VP) selected in etoposide [16] was also grown in AIM-V and showed cross-resistance to etoposide (9-fold), doxorubicin (20- fold), and vincristine (10-fold) (data not shown). These cells have multiple mechanisms of drug resistance, including alterations in topoisomerase II and expression of the membrane pumps, MDR1 and MRP. BEAS-2 normal lung epithelial cells [22] were grown in DMEM-F12 supplemented with 10% fetal bovine serum.

For cytotoxicity testing, extracts were added as indicated to logarithmically growing cells in 1-ml cultures containing 6000 cells/ ml. After 4 days of continuous exposure, cells were counted in a Coulter Z-1 counter. Counts were validated microscopically by hemocytometer after staining with trypan blue. All experiments were done in triplicate and repeated at least three times. The IC50 values, defined as the concentration of extract that had reduced cell counts on day 4 by 50%, were calculated as compared to solvent controls. Mean values were calculated and compared by ANOVA for the three cell lines for that extract.

Cell death assays

The mechanism of cytotoxicity was investigated by the kinetics of cellular DNA fragmentation (Boehringer-Mannheim kit, Indianapolis, Ind.). Briefly, 5·105 cells/ml were incubated for 16 h at 37°C in an atmosphere containing 5% CO2 in AIM-V medium containing 10 lM BudR. The cells were washed and resuspended at 105/ml in BudR-free medium, and 200 ll of this culture was incubated with herbal extract at 2·IC50 for the indicated time, after which 100 ll of the culture medium was removed for quantitation of DNA fragments by ELISA. This is a measure of cell necrosis. DNA fragments within the cells, generated by apoptosis, were measured following cell lysis in bovine serum albumin (BSA)/ Tween 20 at 21°C for 30 min. Following centrifugation, the lysate was used for ELISA.

ELISA was performed in a round-bottomed microtiter plate coated with anti-DNA antibody (mouse anti-human DNA monoclonal, clone MCA-33) overnight at 4°C. Following blocking with BSA, 100 ll labeled DNA fragment solution was added to the coated wells followed by incubation for 90 min at 21°C. The DNA was fixed and denatured by microwave irradiation at 500 W for 5 min. Mouse anti-BudR (mouse monoclonal BMG 6HB) conjugated with peroxidase was added and, following incubation in the dark for 120 min at 21°C, the reaction was stopped by the addition of 500 ll concentrated H2SO4. The resulting color was quantitated by absorbance at 450 nm. Extract-treated samples were compared to untreated controls as background.

Apoptosis was confirmed by TUNEL analysis. Logarithmically growing cells were exposed to herbal extract at 1·IC50 for 3 h. They were then washed twice in phosphate-buffered saline (PBS) containing 1% BSA and resuspended in PBS/BSA at 5·105 cells/ml. To 500 ll cells, 100 ll 4% paraformaldehyde was added and the cells were fixed at 21°C for 1 h. Following washing in PBS, the cells were resuspended in 100 ll cold permeabilization buffer (0.1% Triton X-100, 0.1% sodium citrate) for 2 min at 4°C. The cells were washed twice in PBS and then resuspended in a 50 ll TUNEL mixture containing terminal deoxyribonucleotide- idyl transferase (TdT) and fluorescein-dUTP (Boehringer- Mannheim kit). After incubation in the dark at 37°C for 1 h, the cells were counterstained with propidium iodide and analyzed by flow cytometry.

Gene expression analysis

Cultures (6 ml) of logarithmically growing cells (5·105 cells/ml) were incubated in a medium containing herbal extract at 1·IC50 at 37°C in an atmosphere containing 5% CO2 for 3 h. Following washing twice in PBS, the cell pellets were frozen at –70°C over- night. RNA was isolated by the RNeasy Mini protocol (Qiagen, Valencia, Calif.). Generally, this method yielded 0.5 LG RNA. This amount of RNA was reverse-transcribed to cDNA and labeled with biotin-dUTP, and hybridized to denatured probes to apoptosis- and cell cycle-related genes on filters. Hybridization was detected by binding alkaline phosphatase-streptavidin to the cDNA using a chemiluminescent substrate, CDP-Star (SuperAr- ray, Bethesda, Md.), followed by exposure to X-ray film, generally for 1–2 min.

Control gene targets on each filter included b-actin and GAP- DH as positive controls, and pUC18 as a negative control. The following cell cycle- and apoptosis-related genes were hybridization targets: bad, bcl-2, BCL-w, BCL-x, caspases 1–10, c-myc, E2F, fas, fas ligand, gadd45, iNOS, mdm2, NFkB, p21Waf1, p53, pig7, pig8, Rb, DR5, trail, Casper, CRADD, DAXX, IAP-1, IAP-2, NIK, TNFR2, TRAF2, TRAF3, TRAF5, and DR5. The intensity of hybridization was scored visually in tandem duplicate spots on the filter in relation to the positive controls.

Results

Our initial experiments tested the cytotoxicities of extracts of Chinese herbs on drug-sensitive and drug-resistant SCLC cells, as well as normal lung epithelial cells. As shown in Table 1, the four herbal extracts were cytotoxic at low concentrations. These findings are similar to those found for SPES and PC-SPES in other tumor cell lines [8]. In each case, the IC50 values for multidrug-resistant cells (H69VP) were not different from those for the drug-sensitive cells (H69). Also, the IC50 values for normal lung epithelial cells were significantly higher than those for the tumor cell lines.

We then investigated the mechanism of cytotoxicity using the kinetics of DNA fragmentation as an indication of apoptosis (fragments slowly formed within cells) or necrosis (fragments rapidly formed and released from the damaged cells). Cells were incubated in herbal extract for between 30 min and 8 h, and then the DNA fragments were estimated by ELISA in both the culture medium and cell lysates. Figure 1 shows representative data for H69cells exposed to the four extracts for 90 min. With three of the extracts (SPES, PC-SPES, OLEN), most of the DNA fragments came from within the cells (lysates), indicating apoptosis. However, with GLYC, the fragments were released to the medium from damaged cells, indicating necrosis. Similar results were obtained with H69VPcells(data not shown).

These cytotoxicity experiments were followed byTUNEL assays of DNA breaks in situ. Compared to untreated controls (1–3% apoptotic cells in four experiments), SCLC cells treated with SPES (58–85%), PC-SPES (63–77%), and OLEN (49–81%) showedTUNEL positivity, while cells treated with GLYC did not (1–2%) (Fig. 2). Similar results were obtained withH69VPcells (data not shown).

We analyzed gene transcription in response to herbal extracts using GeneArrays. These arrays have tandemly arranged genes related to the cell cycle and apoptosis. We performed these experiments twice with similar results. Figure 3 shows representative arrayautora diagrams of H69 cells exposed to etoposide orSPES. Our results for H69 cells are summarized inTable 2. While untreated controls had a detectable expression of only a few of the genes studied, cells treated with conventional chemotherapeutic drugs(etoposide and vincristine) showed strong signals for a number of genes involved in the cell cycle regulation and apoptosis. Cells treated with OLEN, SPES and PC-SPES showed a pattern similar to those obtained with the two drugs. In general, cells treated withGLYC did not appear to show this transcription pattern, and instead showed strong expression of the antiapoptosis gene, BCL-2.

Discussion

We undertook these studies for two reasons. First, there is an ancient tradition of Chinese herbal medicine that is based on its own theories and philosophy [2], and we wanted to begin to understand in Western medical terms the effects of the herbal remedies. These herbs are used as mixtures, and while there has been some progress in breaking them down to individual chemical constituents[11], our studies focused on them as they are used in traditional practice. Our second motivation was that many cancer patients use these herbs along with conventional treatments [6, 23], and understanding the cellular effects of the herbs could provide important information for clinical decision-making.

The cytotoxicity data (Table 1) showed that the four herbal extracts were indeed cytotoxic to tumor cells, more so on a dosage basis than to normal lung epithelial cells, indicating some tumor specificity and possibly a favorable therapeutic index. The similarities in IC50 between drug-sensitive and multidrug-resistant cells indicate that these extracts are not affected by the drug resistance mechanisms displayed in H69VPcells, including overexpression of two drug transporters, MDR1 and MRP.

The kinetics of DNA fragmentation showed that extracts SPES, PC-SPES, and OLEN were proapoptotic, while GLYC was pron erotic (Fig. 1). This was confirmed by TUNEL staining in situ (Fig. 2). Most conventional chemotherapeutic drugs are proapoptotic [3], and a group of apoptotic and cell cycle regulatory genes is upregulated in the cells in response to the drugs [24]. We found that the pattern of expression of some of the genes expressed in response to the three pro app to herbal extracts was similar to that observed in these cells after exposure to conventional chemotherapeutic drugs(Fig. 3, Table 2). The response to GLYC alone appeared to be different, at least at the time-point analyzed in these experiments. Interestingly, GLYC is part of PCSPES, which was proapoptotic, while GLYC alone was not. Perhaps other components of PC-SPES are responsible for its proapoptotic activity. While these results do not show how the herbal extracts damaged cells, they suggest that H69 cells respond to the damage at the molecular (transcription) and cellular (apoptosis) levels in a manner similar to their response to chemotherapy.

Our studies indicate the potential usefulness of these Chinese herbal extracts in the treatment of SCLC, particularly drug-resistant disease.

Acknowledgments We thank L. Brown for invaluable assistance with fellow cytometry and R. Lingeman for assistance with molecular biology protocols. The BEAS-2 cells were generously supplied by Dr. I. Laird-Offringa, University of SouthernCalifornia, Norris Cancer Center. SPES and PC-SPES were a giftfrom Dr. S. Chen, New York Medical College. This research was supported by the Pritzker Family Foundation and the W.M. KeckFoundation.

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