Typhonium blumei extract inhibits proliferation of human lung adenocarcinoma A549 cells via induction of cell cycle arrest and apoptosis

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Abstract

Ethnopharmacological relevance

Typhonium blumei Nicolson & Sivadasan is a traditional Chinese medicinal herb endowing with detumescence, detoxification, anti-inflammation activities, and has been used as a folk prescription on anticancer in Taiwan.

Aim of the study

The purpose of this study is to investigate the inhibitory effect of Typhonium blumei (Tb) extract on the viability of different cancer cells and the apoptotic effect of this extract on A549 lung cancer cells.

Materials and methods

Human A549 cell line and other cancer cell lines were treated with different concentrations of Tb extract at different time intervals. Growth inhibition was determined by MTT assay. Apoptosis was detected by cell morphologic observation, cell cycle analysis, and immunoblot analysis on the expression of protein associated with cell death. GC–MS were used to determine the chemical constituents of this extract.

Results

The Tb extract had cytotoxicity toward A549 lung cancer cells (IC50 = 97.7 μg/ml), LNCaP prostate cancer cells (IC50 = 124.5 μg/ml) and MCF-7 breast cancer cells (IC50 = 125.8 μg/ml). Conversely, the adverse effects of Tb extract on normal embryonic lung fibroblast MRC-5 cells (IC50 = 245.5 μg/ml) and embryonic kidney fibroblast HEK293 cells (IC50 = 251.1 μg/ml) were comparatively low. Cytometric analysis results demonstrate that A549 cells were arrested at the G2/M phase by treatment with Tb extract. The extract induced A549 cell apoptosis via the mitochondrial pathway by down-regulating Bcl-2 and Bcl-xL protein expression, up-regulating Bax, Bad and Bak protein expression, and activating caspase-9 and caspase-3. Experimental results of bioactive compound analysis indicate that dibutyl phthalate, α-linolenic acid, phytol, campesterol, stigmasterol and β-sitosterol were the major bioactive ingredients of Tb extract. Although all these compounds had good anti-proliferative effects on A549 cells, campesterol (IC50 = 2.2 μM for 24 h treatment) and β-sitosterol (IC50 = 1.9 μM for 24 h treatment) displayed the greatest inhibitory activity.

Conclusions

Experimental results of this study suggest that the Tb extract exerts potential anticancer activity through the growth inhibition and the apoptosis on A549 cells.

Graphical abstract

Typhonium blumei (Tb) has been used as a folk prescription on anticancer in Taiwan. However, very few studies have investigated its bioactivity scientifically. The results of this study suggest that the Tb extract exerts potential anticancer activity through the growth inhibition and the apoptosis on A549 lung cancer cells.

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Introduction

Some cancer chemotherapeutic agents can abate or reverse cancer development and/or progression. As an important source, plants may produce potential chemopreventive or chemotherapeutic agents. Traditional or folk medicinal herbs have been and are still prescribed to treat many diseases and conditions. Clinical applications have, for many decades, demonstrated their therapeutic effects. Many plant-derived bioactive constituents, including paclitaxel (from Taxus brevifolia), camptothectin (from Camptotheca acuminata), podophyllotoxin (from Podophyllum emody) and vinblastine (from Catharanthus roseus), have been developed as potential sources of anticancer agents (Vickers, 2002, Bosch et al., 2004). Recent scientific efforts have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment. Herb extracts are often administered together with conventional cancer therapies to increase survival rates and enhance the quality of patient life (Kinghorn et al., 2003, Li et al., 2009).

Lung cancer, a common cancer-related death worldwide, ranked first and second in 2009 among the ten leading forms of cancer in Taiwan for both genders, respectively (Department of Health, 2009). More than difficult to cure, lung cancer also has a high incidence of recurrence (Khuri et al., 2001, Li et al., 2004, Molina et al., 2006). The non-small cell lung cancer (NSCLC) constitutes a majority of lung cancers. Either cisplatin or platinum-based chemotherapy is the most common regimen for treating patients with advanced NSCLC. However, these drugs are highly toxic with a low survival profile (De Petris et al., 2006). Cancer cells may resist current chemotheraputic regimens, dominate a cell population and ultimately cause mortality, necessitating the improvment of clinical management against NSCLC. Recently targeting apoptosis, a programmed cell death, is an effective means of treating cancer, including NSCLC (Singhal et al., 2005).

Apoptosis, an important process in cell development and maintenance of tissue homeostasis, plays an essential role as a protective mechanism against carcinogenesis by eliminating damaged cells or abnormal excess cells (Kaufmann and Hengartner, 2001, Schuchmann and Galle, 2004). Apoptosis is characterized by particular morphological changes, including plasma membrane bleb, cell shrinkage, depolarization of mitochondria, chromatin condensation, and DNA fragmentation (Wyllie et al., 1980). The relationship between apoptosis and cancer has been a recent focus. Apoptosis provides a number of useful clues when generating effective therapies, and many chemotherapeutic agents exert their anticancer effects by inducing apoptosis in cancer cells (Schuchmann and Galle, 2004). Therefore, induction of apoptosis has become a principal mechanism by which anticancer therapy is effective (Kundu et al., 2005).

Belonging to the Araceae family (Huang, 2000), Typhonium blumei Nicolson & Sivadasan (formerly known as Typhonium divaricatum (L.) Decne) is a traditional Chinese medicinal herb endowing with detumescence, detoxification, anti-inflammation, antivirus, and anticancer bioactivities. However, its bioactivities have seldom been investigated. Luo et al. (2007) separated lectin, a protein with 48 kDa that can recognize and bind to polysaccharides or glycoproteins expressed on cell surfaces, from Typhonium blumei. This lectin displayed significant antiviral activity against HSV-II of Vero cell line and an anti-proliferative effect on human cancer cell lines, including Pro-01 prostate cancer cells, Lu-04 lung cancer cells and Bre-04 breast cancer cells. In Taiwan, the leaf of Typhonium blumei has been administered as a folk prescription on anticancer (swallow 15 pieces of leaves each time weekly), especially for treating lung cancer and prostate cancer. However, to our knowledge, its anticancer activity has not been verified.

This study investigated how Typhonium blumei (Tb) extract affects cell growth and apoptosis of A549 human NSCLC cells. The anticancer mechanism of Tb extract was also elucidated by analyzing expressions of apoptosis-related molecules, including caspases and Bcl-2 family proteins. Finally, several bioactive ingredients in Tb extract were determined.

Section snippets

Materials and chemicals

The raw materials of Typhonium blumei were bought from a local herb store in Kaohsiung City and were deposited in the Herbarium of I-Shou University (Kaohsiung City, Taiwan). RPMI and DMEM medium were from Gibco (Grand Island, New York, USA). RNase A was purchased from Gentra Systems Inc. (Minneapolis, MN, USA). Annexin V-FITC apoptosis detection kit was obtained from Strong Biotech Co. (Taipei, Taiwan). Antibodies against procaspase-9, caspase-3, -8, -9, Bcl-2, Bcl-xL, Bax, Bad, Bak, and

DNA sequence identification of Typhonium blumei

Plant species are generally categorized based on their appearance, and from tissue slides of flowers, stems, roots, and seeds. However, morphological and histological characterization of herbal medicines often fails to distinguish between closely related species that are not as effective or have potential side effects (Shaw et al., 2002). Sequence comparisons of ITSs are becoming the prevailing method for authentication of medicinal plants at the DNA level; that is, DNA sequence analysis has

Discussion

This study is the first to examine the effect of Tb extract on anti-proliferative activity toward lung cancer cells. Experimental results show that Tb extract had good cytotoxicity against A549 cells, but was less sensitive to normal human cells, including human embryonic kidney cell HEK293 and normal lung fibroblast cell MRC-5, based on the high IC50 value (Table 1). The high sensitivity of malignant cells may in part reflect the difference in growth rates between malignant and non-malignant

Conclusions

This study demonstrates that Tb extract significantly inhibited A549 cell proliferation, while this extract affected the sensitivity of normal lung fibroblast cells to a lesser extent. Treating A549 cells with Tb extract led to a cell cycle arrest at the G2/M phase and induced apoptosis of A549 cells by down-regulating Bcl-2 and Bcl-xL protein expression and up-regulating Bax, Bad and Bak expression. Additionally, dibutyl phthalate, α-linolenic acid, phytol, campesterol, stigmasterol and

Acknowledgements

This work was supported by the National Science Council, Taiwan (NSC 96-2313-B-214-003-MY3) and E-DA Hospital, Taiwan (EDAHI-98003 and EDAHI-99003). The authors thank Prof. Ming-Hong Yen, from Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan, for his assistance to identify Typhonium blumei Nicolson & Sivadasan. Ted Knoy is appreciated for his editorial assistance.

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