Growth Inhibition and Induction of Apoptosis in SHG-44 Glioma Cells by Chinese Medicine Formula “Pingliu Keli”

The present study was carried out to evaluate the effects of the water extract of Chinese medicine “Pingliu Keli” (PK) on human glioma cell viability and apoptosis and to investigate its mechanisms of action in SHG-44 cells. MTT assay showed that PK had a strong cytotoxic effect on SHG-44 cells. The number of live cells was less than 20% after exposure to 90 μg/mL PK for 24 h. PK increased cytotoxicity of SHG-44 cells in a dose-dependent manner. PK caused arrest of SHG-44 cells in G1 phase at low concentration and in G2 phase at high concentration. The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 38% and 52% after treatment with 72 and 108 μg/mL PK, respectively. In addition, PK increased the expression of proapoptotic protein (Bax) and decreased antiapoptotic protein (Bcl-2), with a concomitant increase in the levels of cleaved caspase-3, cleaved caspase-9 and cleaved poly-ADP-ribose polymerase (PARP). These results suggest that PK has a significant apoptosis inducing effect on SHG-44 glioma cells in vitro and caspase-3 may act as a potential mediator in the process.


Introduction
Up to 65% of primary brain tumors are of glial origin and these glial-derived tumors are collectively called gliomas. Glioma is one of the most malignant human tumors [1] and, despite aggressive surgical resection and radiotherapy, the median survival in these patients does not normally exceed one year [1,2]. The use of systemic chemotherapy may improve the efficacy of treatment, but its use is associated with significant toxicity and the long-term prognosis remains poor [3]. It is now being increasingly recognized that intervening critical processes of cancer growth and development with naturally occurring herbal and phytochemical agents to achieve chemoprevention is crucial to decreasing the morbidity and mortality of these and other cancers. Chinese herbal medicine has long been used for treating malignancies [4,5]. Whereas single herbs are seldom used alone, herbal cocktails take advantage of synergy and interactions among a myriad of phytochemicals present in the different herbs to achieve therapeutic efficacy targeting multiple biological and pathological processes while min-imizing side effects [6,7]. However, herbal remedies are yet to be integrated into main stream medicine due to a number of challenges, including herbal standardization and quality control issues, safety and toxicity concerns, interactions with existing therapeutic modalities, a lack of proven efficacy by standard clinical trials and a lack of mechanistic details, to name a few [8,9]. Rigorous in vitro and preclinical animal studies will be essential and necessary to evaluate their efficacy and safety before clinical trials can be contemplated for the chemoprevention and treatment of these major cancers in humans and to transform traditional herbal practices into "evidence-based medicine." In China, the water decoction of "Pingliu Keli" (PK) is employed as a folk remedy for the treatment of glioma [10]. The present study examined the antiproliferative activity of a water extract of PK and its effect on the cell cycle and apoptosis of SHG-44 glioma cells. Furthermore, the levels of several important genes that are strongly associated with the signal transduction pathway of apoptosis were measured to establish the anticancer mechanism of PK.   Figure 1: Inhibition of the cell growth and induction of apoptosis by PK in human malignant glioma cell SHG-44. After treating with different concentration of PK for 48 h, the cells were observed using an inverted microscope (Zeiss Axio Observer A1) or stained with Hoechst 33258, and then photographed with a fluorescence microscope. Compared to the control cells, cells exposed to PK presented typical apoptotic morphology with cell shrinkage, nuclear condensation, and formation of apoptotic bodies, with rupture of cells.

Cell Morphological Assessment Effect of PK on Cell
Morphology. Differences in cell morphology were observed between PK-treated and control cells by light microscopy. The most conspicuous changes observed in PK-treated cells included cell shrinkage and extensive detachment of the cells from the cell culture substratum. These changes, which were characteristic of cell apoptotic death, became visible after 12 h of PK treatment, but were absent in control cells. The morphological changes became more remarkable with increased time of drug treatment. These observations suggested that cells treated with PK detached from the substratum and died by apoptosis (Figure 1(a)). The occurrence of apoptosis was further verified by Hoechst staining, which detects chromatin condensation, one of the hallmarks of apoptotic cell death. Some differences were observed in the nuclei of PK-treated anduntreated SHG-44 cells after staining with Hoechst 33258 (Figure 1(b)). The Hoechst 33258 dye stained morphologically normal nuclei dimly blue whereas PK-treated cells demonstrated smaller nuclei with brilliant blue staining. The changes in nuclear morphology were initially observed after 24 h of PK treatment and increased thereafter. These results demonstrate that PK induces morphological changes characteristic of apoptotic cell death.

Effects of PK on the Cell Proliferation of SHG-44.
To test the effect of PK on the proliferation of SHG-44 cells, the cells were treated with different concentrations of PK. After 48 h incubation, the cell viability was measured by MTT assay. PK treatment significantly inhibited the growth of SHG-44 cells, the number of viable cells decreases as the concentration of PK increases ( Figure 2). The effect of PK treatment is statistically significant when compared with the control group (P < .05).

Different Effects of Low and High Concentration of PK on SHG-44 Cell
Cycle. Analysis of cell-cycle phase distribution was carried out to study the antiproliferative mechanism of PK. Different concentration of PK has different effects on SHG-44 cell-cycle. At lower doses of PK (72 μg/mL), the number of SHG-44 cells during G0/G1 was increased, and the S phase was reduced dramatically. However, when the concentration was up to108 μg/mL, most cells were arrested at G2/M phase. The cell-cycle arrest was evident at 24 h treatment, and many cells died after 36 h treatment of high concentration of PK. These results indicated that PK could inhibit SHG-44 cells synthesizing DNA at low concentration, whereas high concentration of PK induced G2 arrest by inhibiting DNA synthesis and cell division (Figure 3).

Quantification of Apoptosis by Flow Cytometry with
Annexin V/PI Staining. Additional evidence for the occurrence of apoptosis was obtained by double staining of the cultures with propidium iodide (which stains the nuclei of dead cells) and annexin V-FITC, a protein that binds with high affinity to phosphatidylserine, which is translocated  from the inner to the outer membrane leaflet early in the apoptotic process. Control cells stained negative for both propidium iodide and annexin V-FITC. PK-induced cells, on the other hand, showed many annexin V-positive cells.
The majority of the cells were negative for propidium iodide, indicating that they were at an early stage of apoptosis. The double positive staining of particular cells revealed that these cells were at a late apoptotic (or necrotic) stage. Take together, these findings provide strong evidence that PK induced cell death through apoptosis (Figure 4).

Western
Blot Analysis of PARP, Caspase-3, Caspase-9, Bax, and Bcl-2/X L Protein. After exposure with PK at different concentration for 48 h, total cell lysate was prepared and an equal amount of protein was subjected to SDS-PAGE. Western blot analyses were done with anti-PARP, -caspase-3, -caspase-9, Bcl-2, Bcl-X L , Bax, and GAPDH primary antibodies as described in materials and methods. PK increased the expression of proapoptotic protein (Bax) and decreased antiapoptotic protein (Bcl-2, Bcl-X L ), with a concomitant increase in the levels of caspase-3, caspase-9 and cleaved poly-ADP-ribose polymerase (PARP) in SHG-44 after treatment for 48 h ( Figure 5). These data showed that PK induced SHG-44 cells apoptosis through a caspase dependent pathway ( Figure 6).

Discussion
Malignant gliomas are the most common primary brain tumors. The patients with malignant gliomas remain  poorly responsive to multimodality therapeutic interventions, including surgery, radiotherapy, and chemotherapy [11,12]. It has been reported that tumor growth is dependent on not only the rate of cellular proliferation, but also that of cell death [13]. Dysregulation of apoptosis contributes directly to tumor development and progression including brain tumors [14][15][16]. In addition, neoplastic cells' loss of the ability to undergo apoptosis is an important factor that determines the response to treatment with radio-or chemotherapy [17,18]. SHG-44 human glioma cell line was derived from a patient diagnosed with a most malignant and highly invasive grade IV glioma, known as GBM, and is frequently used as a model system to study invasive properties of gliomas in many studies. In the present paper, we demonstrated that PK inhibited the glioma tumor growth and induced the apoptosis of SHG-44 glioma cells. Hoechst 33258 staining and FACS analysis demonstrated that the majority of the glioma cell death is due to apoptosis, and the difference between cell death and apoptosis may result from nonspecific cell death. To gain insight into the molecular mechanism involved in apoptosis by PK, expression of apoptotic-related proteins, Bcl-2, Bcl-X L , Bax, and caspase-3, were assessed in SHG-44 glioma cells. We showed that the induction of apoptosis was accompanied by the down-regulation of Bcl-2 gene expression and up-regulation of Bax gene expression, suggesting that alteration of Bcl-2 and Bax is directly or indirectly involved in the apoptotic effect of PK in SHG-44 cells. The expression ratio of proapoptotic and antiapoptotic genes may determine whether a cell lives or dies following an insult [19]. Overexpression of Bcl-2 protects cells from apoptosis following exposure to a number of different proapoptotic stimuli [20], whereas overexpression of Bax renders cells more sensitive to proapoptotic stimuli [21]. Similarly, we also found that PK treatment causes a decreased Bcl-2 expression and increased level of Bax, which may be responsible for the induction of apoptosis in SHG-44 glioma cells. It has been reported that the ICE/caspase family plays Caspase dependent cell death Figure 6: Regulation of the intrinsic apoptotic pathway by PK. The active compounds of PK reduced their effectiveness after digesting and absorbing by patients. PK increased the expression of proapoptotic protein (Bax) and decreased antiapoptotic protein (Bcl-2, Bcl-X L ), with a concomitant increase in the levels of caspase-3, caspase-9 and cleaved poly-ADP-ribose polymerase (PARP) in SHG-44 cells. Caspase dependent cell death was the possible mechanism of PK's antitumor activity.
a crucial role in apoptosis [22]. In particular, caspase-3 has been shown to be a key component of the apoptotic machinery [23][24][25]. Our data demonstrated that caspase-3 was activated by PK, indicating that caspase-3 might be one of the critical steps in PK-induced apoptosis.

Conclusion
This study clearly demonstrates that the water extract of PK strongly inhibits cell proliferation and induces apoptosis in SHG-44 cells. PK induced apoptosis through the activation 8 Evidence-Based Complementary and Alternative Medicine of caspases-9 and -3 and degradation of PARP. Because apoptosis was regarded as a new target in discovery of anticancer drugs, these results confirm the potential of PK as an agent of chemotherapeutic and cytostatic activity in human glioma cells. Although PK is most commonly used in Chinese medicine, the mechanistic aspects of its effects are still unknown and potential of its bioactive components are yet to be recognized [25,26]. Thus, evaluation and characterization of the water-soluble active components for discovery of potentially safe glioma-therapeutic phytoreagents is warranted.