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Journal of Cancer Research and Therapeutics, Vol. 7, No. 4, October-December, 2011, pp. 421-426 Original Article Pro-apoptotic effects of Paecilomyces hepiali, a Cordyceps sinensis extract on human lung adenocarcinoma A549 cells in vitro Asmitananda Thakur1, Ren Hui1, Zhang Hongyan2, Yang Tian1, Chen Tianjun1, Chen Mingwei1 1 Department of Respiratory Medicine, The First Affiliated Hospital of Xian Jiaotong University, School of Medicine, Xian-710 061, People's Republic of China Code Number: cr11114 DOI: 10.4103/0973-1482.92007 Abstract Background: Paecilomyces hepiali (PH) is a derivative of Cordyceps sinensis (CS), a fungus that has been shown to have anti-cancer and pro-apoptotic effects. Here, we aimed to investigate the effect of in vitro PH treatment on cell proliferation, cell cycling, apoptosis, and tumor necrosis factor-alfa (TNF-α) mRNA expression in human lung adenocarcinoma A549 cells (A549). Keywords: Apoptosis, Cordyceps sinensis, Paecilomyces hepiali, Tumor necrosis factor-mRNA Introduction Primary neoplasm of the lung is a key health concern with a dismal prognosis. The most common type arising in non-smokers, women, and young adults (<45 years old) is adenocarcinoma. [1] Development of lung cancers such as adenocarcinomas is a multi-step process which involves carcinogens and tumor promoters. Deregulation of cellular homeostasis and promotion of malignancy are driven, in large part, by cancer cells' ability to evade apoptosis. [2] Because of the complexity of genetic lesions and the variation in derivative phenotypic changes of cancer cells, multifunctional drugs that affect tumor cells at diverse levels must be used for treatment. Unfortunately, cancer cells can mutate and become resistant to available drugs, which has spurred interest in the adjuvant use of natural bioactive compounds in conventional chemotherapy. [3] This interest in natural compounds is certainly valid, as approximately one-half of the anti-cancer drugs developed since 1960 are derived from plants. [4] One potential cancer treatment derives from Cordyceps sinensis (CS), a fungus endemic to Himalayan alpine habitats (at an elevation of 3600-5000 m). This species, which is endoparasitic on caterpillars, has long been used for medicinal purposes in a variety of Asian countries, including India, China, Nepal, and Japan. [5] It has been shown to function as an aphrodisiac, [6] an analgesic, [7] an immune modulator, [8] a free radical scavenger, [9] and an anti-cancer agent. [10],[11],[12] Perhaps most intriguing are reports that extracts of this fungus promote apoptosis. [13],[14],[15],[16],[17],[18],[19] The mechanisms behind the anti-cancer and immune-modulating activities after treatment with CS are attributed to the production of polysaccharides, sterols, lipids, nucleosides and deoxy-nucleosides from cells. [20],[21],[22] CS derivatives have been produced via aseptic mycelia cultivation, the two most well-studied of which are Paecilomyces hepiali (PH; strain CS-4) and Cephalosporium sinensis.[5] Experiments have shown that PH can inhibit tumor proliferation, invasion, metastasis, and neovascularization, induce apoptosis, reverse drug resistance, enhance immunity, and protect hepatic function. [9],[14] Polysaccharide factions of CS have been found to augment triptolide-induced apoptosis in cultured leukemia HL-60 cells; [23] furthermore, aqueous extracts of CS induce apoptosis through a signaling cascade of death receptor-mediated extrinsic and mitochondria-mediated intrinsic caspase pathways. [15] These results suggest that CS derivatives could have anti-cancer activity, though, to our knowledge, this has not yet been fully investigated; specifically, no research has examined the potential antitumor effects of PH on the human lung adenocarcinoma A549 cell line. In this paper, we targeted human lung adenocarcinoma A549 cells with an aqueous extract of PH (in vitro) and quantified resulting changes in cell growth, cell cycle, rate of apoptosis, and TNF-α mRNA expression. Materials and Methods The human lung adenocarcinoma A549 cell line was supplied from the Chinese Academy of Sciences (Shanghai Institute for Biological Sciences Cell Bank, China), and preserved at the Central Laboratory. PH standard powder (100% purity, 4 g/package) was purchased from Zhejiang Wanfeng Medicines Group Co. Ltd., China (HS code: 30029090, United States Food and Drug Administration registration number: 17217333984). Calf serum was purchased from Lanzhou Minhai Biological Co. (China). RPMI-1640 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ethidium bromide (EB), and agarose were bought from Sigma Co. (St. Louis, Missouri, USA). The Annexin V-FITC apoptosis detection kit and FACS Calibur Flow Cytometer were purchased from Becton Dickinson (Franklin Lakes, New Jersey, USA). The RT-PCR kit was purchased from Fermentas MBI (Maryland, USA). All other reagents were purchased from Sigma Co., and all other chemicals were purchased from Aldrich Co. (Steinheim, Germany). To prepare the PH solution, 4 g of PH powder was soaked overnight in 10 times the volume of distilled water. The following morning, an ultrasonic extraction was performed for 20 min at 60°C. The extract was then centrifuged at 7200 rpm for 15 min and the supernatant discarded. The residue was added to 10 times the volume of distilled water (pH adjusted to 7.5) and ultrasonic extraction was performed for 20 min at 80°C. The extract was then centrifuged at 7200 rpm for 15 min and the supernatant discarded. The residue was freeze-dried and dissolved in 40 ml of phosphate buffer saline (PBS), creating a crude drug concentration of 100 mg/ml, pH adjusted to 7.0. A 0.45-μm membrane filter was used for removal of bacteria and the final solution was preserved at -20°C. RPMI-1640 medium was added to obtain the required concentration. A549 cells containing 10% calf serum, RPMI-1640 culture medium at 37°C were incubated in 5% CO 2 incubator for culture and growth in 96 well plates (each with cell suspension of 5.0 × 10 3 ), and final volume of 200 μl/well. After 24 h of culture the original culture medium was discarded and replaced with PH diluted with RPMI-1640 to concentrations of 0.25, 0.50, 1, 2, and 4 mg/ml. A control (no drug intervention) and a blank (medium only) were also prepared (each repeated in four wells) and each group was cultured for 24, 48, and 72 h respectively. Cells in each group were washed twice with warm PBS, after which 180 μl of serum-free medium was added to the wells. Each well was also treated for 4 h with 20 μl of MTT (5 mg/ml); the MTT assay is a calorimetric assay used to assess the viability (cell counting) and proliferation (cell culture) of potential medicinal agents. Finally, the medium was discarded, 150 μl of dimethyl sulfoxide (DMSO) was added to each well, and the cells were subjected to 10 min of vortex oscillation. A flow cytometer was used to observe the optical density (OD) value at an excitation wavelength of 490 nm. Data collection and processing were performed with CellQuest software, and the following formula was used to calculate cell proliferation: Cell proliferation (%) = [1- (OD of drug-treated samples - OD of blank sample) / (OD of control - OD of blank)] × 100 %, where ≥50 % inhibition indicated that the cells were highly sensitive, 31-49% inhibition indicated that the cells were moderately sensitive, and ≤30% inhibition indicated that the cells were non-sensitive to the drug treatment. Logarithmic growth phase of A549 cells cultured in 0.5, 1, and 2 mg/ml PH solution for 24 h were washed and fixed overnight with 5 ml of 70% ethanol. The cell suspension was centrifuged at 1000 rpm for 10 min at 4°C, after which the ethanol was discarded. The suspension was rewashed using 1 ml of cold PBS and centrifuged at 1000 rpm for 10 min at 4°C. When cells had reached the dark phase, 30 μl of Propidine iodide (PI) (0.5 mg/ml) and 3 μl of RNase A (10 mg/ml) were added to 400 μl of cell suspensions (4°C, 30 min). Flow cytometry (FCM) analysis was used to observe percentage distribution of A549 cells in the different cell cycle stages. A549 cells cultured in PH solution for 24 h were digested and centrifuged at 1000 rpm for 5 min, after which a 10 3 cell/ml suspension was prepared. In a 5-ml flow tube, 100 μl of cell suspension was added to 5 μl of Annexin V-FITC (a sensitive and specific detector of apoptosis) and 10 μl of PI, then mixed for 15 min in the dark phase at room temperature. Next, 400 μl of PBS was added to the solution. An FCM detector was used to count cells (1 × 10 3 ) at an excitation wavelength of 490 nm. CellQuest software was used for data collection and processing. An RNA fast 200 kit was used to extract the total RNA, after which the random primer method cDNA two-step reverse transcription kit was used, as per instructions in the Fermentas MBI manual. After RT-PCR amplification and electrophoresis, post-transcriptional primers were analyzed using BandScan software. TNF-α, β-actin, and TNF-α/β-actin electrophoretic band gray values (which represented the relative expression of TNF-α mRNA frequency) were measured for each group. Statistical analysis Five replicates were performed for each experiment, and data were averaged and presented as mean ± standard deviation [X ± S]. A single-factor analysis of variance (ANOVA) was used for comparison between the groups. SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for data processing. Significance was defined as P < 0.05. Results After 24 h of culture, the control group had spindle-shaped cells, uniform growth, normal morphology, and active proliferation; PH intervention groups had decreased cell density, round, swollen cell morphology, and increased cytoplasm. Variations in cell morphology were concentration-dependent [Figure - 1]. At 24 h, cells were not sensitive to the 0.25 mg/ml intervention, were moderately sensitive to the 0.5 mg/ml and 1 mg/ml interventions, and were highly sensitive to the 2 mg/ml and 4 mg/ml interventions, which strongly inhibited cell proliferation. The 0.25 mg/ml intervention had a similar effect at both 24 and 48 h (P > 0.05), but inhibited cell proliferation significantly more at 72 h (P < 0.05). Cells were sensitive to the drug interventions in a positive time- and concentration-dependent manner (both P < 0.05) [Table - 1]. After 24 h of drug intervention, cells in the PH intervention and control groups were distributed significantly differently among the phases of the cell cycle (P < 0.05). The drug intervention group had an increased proportion of cells in the G0/G1 and G2 phases, and significantly fewer cells in the S phase (P < 0.05; [Table - 2], [Figure - 2]). A variety of cell types were observed after 24 h of PH intervention treatment: surviving cells, Annexin-V (-), PI (-); cells with early apoptosis, Annexin-V (+), PI (-); and cells with late apoptosis, Annexin-V (+), PI (+). The rate of apoptosis varied depending on the concentration of the PH intervention; the rate was significantly higher in the drug intervention groups than in the control group and increased as drug concentration increased (P < 0.05; [Table - 3], [Figure - 3]). Two mg/ml PH intervention had a 29.22 ± 0.42% apoptosis rate. TNF-α mRNA expression was higher in the PH intervention groups than in the control group and was PH concentration-dependent (P < 0.05; [Table - 4], [Figure - 4]). Discussion Existing cancer therapies have been associated with subclinical damage that may only be recognized in the presence of a secondary inciting factor. Standard anticancer chemotherapeutic strategies are often limited by mechanisms of resistance encountered in tumor cells, [24] as well as by patients' tolerance to drug toxicity. [2] The challenge for clinicians is to preserve or augment the cancer cure rate while decreasing the risk of serious treatment-related complications. Adjuvant use of bio-products became trendy in medical practice because of their tumor specificity and novel chemical properties which counter these side-effects. [3] CS and its derivatives are strong inhibitors of IκBα degradation and moderate inhibitors of IκBα phosphorylation. [16] All these properties together with recent advancement in fermentation, isolation and structure elucidation technologies have made research into CS derivatives seem worthwhile. In this study we found that PH intervention caused decreases in total cell numbers 24 h after treatment, and that this decrease occurred in a dose-dependent manner. Further, cells in the drug intervention group had distorted cellular morphologies, a characteristic that also showed dose dependence. The cell cycle consists of a definable sequence of events that characterize the growth and division of cells. It has its own internal control, called checkpoints. These checkpoints induce arrest or delay of cell cycle progression and provide sufficient time for DNA repair, if possible, thereby protecting the cells from propagating damaged DNA. Although the mechanisms to sense DNA damage remain to be fully understood, it has been shown that double-stranded DNA breaks activate the Ataxia telangiectasia mutated (ATM) and ATM rad 3-related ( ATR) kinases, which play central roles in the DNA damage response by phosphorylating several key molecules, including CHK1, CHK2, p53, BRCA1, and NBS1. This in turn leads to further downstream signaling and cell cycle arrest or delay at G 1 , S, and G 2 -phase. The results of our MTT and FCM assays examining the effect of PH on cell cycle intervention were in agreement with previous findings on studies examining the effects of CS extract on cellular proliferation; [16],[25],[26],[27] specifically, cellular proliferation was inhibited and cell cycle was arrested in a dose-dependent manner. Here, the anti-proliferative potential of PH was highlighted by the increased proportion of G0/G1- and G2-phase cells and the decreased proportion of S-phase cells. A previous study found that mycelium polysaccharide produced by C. jiangxiensis arrested mitosis and promoted apoptosis, indicating that it possessed potent antitumor capabilities. In the current study, we found higher rates of apoptosis among cells treated with PH, a reaction that occurred in a dose-dependent manner; these results agreed with earlier findings. We also observed a dose-dependent increase in TNF-α mRNA expression after 24 h of intervention with PH, which may explain the increased levels of apoptosis detected in the drug intervention groups. The overwhelming functions of TNF-α in immune modulation are well documented. It is known that Tumor necrosis factor receptor type 1- associated death domain protein (TRADD) binds Fas-associated protein with death domain (FADD), which then recruits the cysteine protease caspase-8. A high concentration of caspase-8 induces its autoproteolytic activation and subsequent cleaving of affector caspases, leading to apoptosis. It can be argued that NF-κβ enhances the transcription of C-FLIP, Bcl-2, and cIAP 1/cIAP 2, inhibitory proteins that ultimately interfere with the apoptotic signaling; however, activated caspases cleave several components of the NF-κβ pathway, including RIP, IKK, and the subunits of NF-κβ itself. We believe this may be a possible explanation to the significant correlation between increased TNF-α mRNA expression and higher apoptosis rates at 24 h of PH intervention. Total cell density is an indicator of cell proliferative activity and of cytotoxic effects of the extract; the results of this study reinforce the relationship between apoptosis in human lung adenocarcinoma line A549 cells and the concentration of in vitro PH intervention. Various bioactive components from CS have been reported. [28],[29],[30] These include cordycepin and anti-tumor adenosine derivatives. Recent reports have indicated that CS contains polysaccharides exhibiting anti-oxidant activity and nucleosides that inhibit platelet aggregation. [31] The bioactive compounds involved in the activities claimed include polysaccharides, modified nucleosides, and cyclosporine-like metabolites which are produced by this fungus-related species. [32],[33],[34] More mechanism-based, disease-oriented pharmacological studies are required to determine the active chemical constituents. There are some limitations to this study. For instance, we did not measure a secondary marker of apoptosis, such as levels of cleaved PARP (poly-ADP-ribose polymerase) or (Adenosine triphosphate) ATP (since exhaustion of ATP shifts the cell from apoptosis to necrosis). We also did not identify potentially active compounds using methods such as amplified fragment length polymorphism (AFLP)-based DNA fingerprinting. However, we believe that the results of this study provide an excellent baseline for future research effort involving multiple cell lines and in vivo study. Conclusions Cumulatively, our findings show that PH inhibits cell proliferation, causes cell cycle arrest, and induces apoptosis of A549 human lung adenocarcinoma cell lines in vitro and in a dose-dependent fashion. Further studies on a normal cell line and other lung cancer cell lines are required to establish the potential of PH in the treatment of lung adenocarcinoma. PH represents an immense source of bioactive compounds that may make them very promising natural agents for in vivo studies on their immune-stimulating and anti-cancer properties. We believe this research will provide insight for further research based on the application of these naturally occurring compounds. Acknowledgment This research was supported by a grant from the National Nature Science Foundation of China (grant number-30672658) for scientific development. The authors were equally involved in the research and preparation of this manuscript. References
Copyright 2011 - Journal of Cancer Research and Therapeutics The following images related to this document are available:Photo images[cr11114f4.jpg] [cr11114f1.jpg] [cr11114f3.jpg] [cr11114t2.jpg] [cr11114t1.jpg] [cr11114t3.jpg] [cr11114f2.jpg] [cr11114t4.jpg] |
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