Morphological Study on Hela Cells Apoptosis Induced by Lycium barbarum Polysaccharides

ZHU Cai-ping，ZHANG Sheng-hua，XIAO Jun-xia

(1. College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062, China；2. College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China)

Morphological Study on Hela Cells Apoptosis Induced by Lycium barbarum Polysaccharides

ZHU Cai-ping1，ZHANG Sheng-hua2，XIAO Jun-xia2

(1. College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062, China；2. College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China)

Lycium barbarum polysaccharides (LBP), isolated with boiling water from Lycium barbarum fruits, a famous Chinese medicinal herb, is one of the most important functional constituents in Lycium barbarum. In this study, the effect of LBP at the range of 3.125 to 200 mg/L on the proliferation of Hela cells was measured. After Hela cells were treated with LBP, typical apoptotic morphological changes were observed by fluorescence microscope, transmission electron microscope (TEM) and laser scanning confocal microscope (LSCM). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay also confirmed that LBP-treated Hela cells showed apoptotic features. The results suggest that LBP is a potential antitumor compound.

Lycium barbarum polysaccharides；human cervical carcinoma Hela cells；apoptosis；morphology

Cancer is a disease state caused by the disruption of cellular homeostasis between cell death and cell proliferation[1]. For a long time, the proliferation of neoplastic cells was considered the major marker of tumor progression and prognosis. In fact, the growth of malignant tumors depends on the proliferation and death rates of cancer cells[2]. Apoptosis, a major process of programmed cell death, plays an important role in the regulation of tissue development and homeostasis[3-5]. Induction of apoptosis has been an important approach in cancer therapies. In recent years, there has been a global trend toward the use of natural substances present in herbs as anticancer foods. Many studies have demonstrated that many natural products isolated from plant sources possess antitumor properties[6].

Lycium barbarum polysaccharides (LBP), extracted from Lycium barbarum that is a kind of traditional Chinese herb, has many bioactivities such as anti-peroxidation, hypoglycemic, hypolipidemic and immunological activities[7-12]. Recently, it was reported that the LBP-standardized Lycium barbarum fruit juice GoChi might support the body health by increasing endogenous factors, such as SOD and GSH-Px, reducing the MDA level and protecting membranes fromoxygen radical-mediated damage[13]. In addition, many experiments have showed that LBP has anticancer effect both in vivo or in vitro, for example, LBP can inhibit tumor growth, protect thymus gland cells and decrease the levels of serum VEGFTGF-β 1 in H22-bearing mice[14]. LBP at the dose of 5－40 mg/L can up regulate the levels of IL-2 and TNF- mRNA in human peripheral blood mononuclear cells[15]. LBP at 20－1000 mg/L can inhibit the growth of human leukemia HL-60 cells in dose-dependent manner and reduce the membrane fluidity[16]. LBP at 100 mg/L can inhibit the proliferation of human hepatoma QGY7703 cells, induce the cell cycle arrest and increase the intracellular calcium in apoptotic system[17].

Cervical carcinoma is one of the most common malignant tumors in the women procreation system, with a combined worldwide incidence of almost half a million new cases annually, second only to breast cancer[18]. Therefore, it ,s important to seek a chemotherapeutic/chemopreventive agent against cervical carcinoma to reduce the incidence and mortality. It s re,ported that LBP can activate macrophages, affect the synthesis of DNA of U14cervix cancer cells in mice[19], and LBP could increasingly inhibit the growth of cervical carcinoma Hela cell line with dose-effect. The biggest inhibition rate was up to 96.85% in those treated with LBP, and the apoptosis rate was 36.8%[20]. In this study, the effect of LBP on proliferation in Hela cell line were tested, and to discuss the mechanism of growth inhibition in Hela cells, the morphological changes were observed by inverted microscope, fluorescence microscope, laser scanning confocal microscope (LSCM) and transmission electron microscope (TEM). Moreover, TUNEL assay was also conducted to determine the apoptosis index.

1 Materials and Methods

1.1 Materials, reagents and instruments

Human cervical carcinoma cell Hela line was supplied by China Center for Type Culture Collection (CCTCC). Dried fruits of Lycium barbarum harvested in Ningxia province were purchased in a local supermarket.

Trypsinase and RPMI-1640 were obtained from GIBCO BRL (Grand Island, NY, USA). Penicillin and streptomycin were produced by North China pharmaceutical Group Corporation. Dimethyl Sulphoxide (DMSO), acridine orange (AO), [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] (MTT) and ethidium bromide (EB) were obtained from Sigma Chemical Corporation. Newborn bovine serum (NBS) was obtained from Wuhan Sanli Biotechnology Corporation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System Detection Kit and phosphate-buffered saline (PBS, pH7.2－7.6) were purchased from Wuhan BOSTER Biotechnology Technology Ltd.

Thermo electron Multiskan MK3 ELISA reader (USA). Leica DFC300FX fluorescence microscope (Germany). Leica TCS SP2 AOBS MP LSCM (Germany). Hitachi H-600 TEM (Japan). Ultrotome NOVA LKB ultramicrotome ( Broma, Sweden).

1.2 Preparation of LBP

Lycium barbarum fruits were dried at 60 ℃ and ground to fine powder. The ground powder samples were refluxed to remove lipids with chloroform:methanol solvent (2:1) (V/V). After filtering, the residues were air-dried, and then refluxed again with 80% ethanol at 80 ℃ to remove oligosaccharides. The residues were extracted four times in boiled water and filtered. The combined filtrates were concentrated by a rotavapor at 60 ℃, and then precipitated using 95% ethanol, 100% ethanol and acetone, respectively. After filtering and centrifuging, the precipitate was collected and vacuum-dried. According to GC analysis, the dried LBP was composed of rhamnose, arabinose, xylose, mannose, glucoseand galactose[21]. LBP was prepared as a stock of 1000 mg/L in basal medium RPMI-1640 and kept at －20 ℃. For all tests, final concentrations of the tested compound were prepared by diluting the stock with RPMI-1640. Control cultures received the same volume of RPMI-1640.

1.3 Cell line and culture

Human cervical carcinoma cell Hela line was maintained in monolayer culture at 37 ℃ and 5% CO2in RPMI-1640 supplemented with 10% inactivated NBS, 10 U/mL penicillin, 10 mg/mL streptomycin.

1.4 Cell proliferation assay

Suppression of cell proliferation by LBP was measured by MTT assay[22]. The Hela cells were incubated in 96-well culture plates (2.0×104cells per well). After 24 h incubation, the cells were treated with LBP (0, 3.125, 6.25 , 12.5, 25, 50, 100, 200 mg/L) for 2, 4 and 6 days. The culture medium was replaced every other day. MTT solution (5 mg/mL) were added to each well and incubated at 37 ℃ for another 4 h. After adding stop solution DMSO (150 μL/well), the absorbance at 490 nm was measured on an ELISA reader. The percent viability of the treated cells was calculated as follows: A490nmsample/ A490nmcontrol×100%.

1.5 Morphological observation

Hela cells were cultured with basal medium and medium contains 6.25 mg/L LBP for 4 days. Then they were examined with fluorescence microscope, LSCM and TEM. The cells were cultured without and with 6.25 mg/L LBP for 4 days. At the end of the treatment, the cells were washed twice with PBS, trypsinized, centrifuged, and then resuspended in culture medium, respectively. 95μ L of cell suspension was then mixed with 5μ L of the dye mixture, containing 100 mg/L AO and 100 mg/L EB in PBS. After staining, cells were visualized immediately under fluorescence microscope. The cells were dyed by acridine orange before examined by LSCM. Before observed by TEM, the monolayer cells were detached by tryplsinization and washed twice with PBS and cell pellets were fixed with 2.5% glutaraldehyde for 2 h at 4 ℃ and then incubated with 1% osmium tetroxide (OSO4) for 1 h at 4 ℃. After dehydration in a serie concentration of ethanol and infiltration in propylene oxide, cells were embedded in Epon 812. Ultrathin sections (60 nm), obtained with an LKB ultramicrotome were stained with uranyl acetate and lead citrate, then cell morphology was observed by TEM at 80 kV.

1.6 TUNEL assay

Apoptosis index (AI) was estimated using the TUNEL assay. After the Hela cells were incubated for 4 days with the medium containing 6.25, 25 mg/L and 100 mg/L LBP, the cells were fixed with 4% Paraformalclehyde for 30 min at room temperature, followed by washing in PBS and distilled water twice (2 mins each time), respectively. Then, the cells were stained with TUNEL assay kit according to the manufacturer ,s instructions. A negative control, without the addition of TdT enzyme, was included in each experiment. Finally, the cells were slightly counterstained with hematoxylin. The cells were observed by light microscope, dark brown DAB signals indicated positive stained cells and blue shades signified unreacted cells, and the apoptosis index was calculated as follows: (the number of TUNEL reactive nuclei/total number of cells counted) ×100%[23].

1.7 Statistical analysis

The results were expressed as mean ± standard deviation. The difference between control and LBP treated cells was evaluated using Student ,s t-test. P value less than 0.05 was considered statistically significant.

2 Results and Analysis

2.1 Effects of LBP on Hela cells proliferation

Hela cells were cultured in 10% NBS-containing medium with or without LBP (0, 3.125, 6.25, 12.5, 25, 50, 100 mg/L and 200 mg/L) during 6 days and cell proliferation evaluated by the MTT test. Under the same condition treated by LBP (3.125－200 mg/L), a dramatic decrease in proliferation was observed until 4 days (Fig. 1), especially at 4 days for 6.25 mg/L LBP where the percentage of inhibition was 35% (P＜0.05), which showed that LBP at the dose of 6.25 mg/L had obviously cytotoxic effect on Hela cells.

Fig.1 Effect of LBP on Hela cell proliferation

2.2 Morphological observation by fluorescence microscope

Fig.2 Morphological observation of Hela cells with AO/EB double staining by fluorescence microscope

To determine whether the cytotoxic effect of LBP was related to the induction of apoptosis, morphological assay of cell death was investigated by using the AO/EB double staining for fluorescence microscopy. This method combines the differential uptake of fluorescent DNA binding dyes AO and EB and the morphologic aspect of chromatin condensation in the stained nucleus, allowing one to distinguish viable, apoptotic, and necrotic cells. Viable cells possess uniform bright green nucleus. Early apoptotic cells show bright green areas of condensed or fragmented chromatin in the nucleus, and necrotic cells show uniform bright orange nucleus. As shown in Fig. 2, no obvious morphological changes wereobserved in the control group (Fig. 2A); however, Hela cells exposed to LBP at the dose of 6.25 mg/L for 4 days exhibited condensed chromatin, fragmented nuclei and appearance of apoptotic bodies (Fig. 2B).

2.3 Morphological observation by LSCM

In order to further identify apoptotic cells, LSCM was used with AO staining in this study. Excited by light at the wavelength of 460 nm, RNA in cytoplasm and nucleolus appeared red while DNA in nucleus was green observed under the excitation wavelength of 502 nm. As shown in Fig. 3, after the cells were cultured for 4 days, the nucleus showed bright green and the cell cytoplasm showed dark green, which suggested that the treatment of LBP(6.25 mg/L) induced the apoptosis. In control cells, the nucleus were bigger and smoother than that of cells treated by LBP, while among LBP treated cells, many, , apoptoticwere observed. The compact masses of chromatin aggregated along the nuclear membrane. Round, compact granular masses appeared near the center of the nucleus and there was a reduction in nuclear volume. At the same time the cytoplasm displayed condensation. Some of the nucleus degenerated into discrete spherical or ovoid fragments of highly condensed chromatin.

Fig.3 Morphological observation of Hela cells by LSCM

2.4 Morphological observation by TEM

To further characterize the morphologic changes in the infected cells, we performed the TEM analysis (Fig. 4). The untreated Hela cells have the normal morphology, abundant microvilli around the cell membrane, all the organelles keep integrity, nuclear membrane was smooth (Fig. 4A), plenty of mitochondrial in the cell (Fig. 4B). After treatment with 6.25 mg/L LBP for 4 days, the reduced cell volume and shrank cytoplasm was observed under TEM, but plasma membrane remained well defined. Condensed chromatin located along nuclear envelope and formed irregularly crescents shape at the nuclear edge. Nuclear membrane became irregular, Microvilli decrease (Fig. 4C), and large lipid droplets and increasing larger lysosomes in cytoplasm could be observed (Fig. 4D). All of the induced changes are the typical indications of apoptosis.

Fig.4 Morphological observation of Hela cells by TEM

2.5 Detection of apoptosis by TUNEL assay

To determine the effect of LBP on apoptosis, Hela cells were measured by TUNEL method after incubating in the presence or absence of LBP medium for 4 days. The TUNEL signal, as an apoptosis marker, appeared as dark brown nuclei (Fig.5B, C, D), while the nuclei was dark blue in the control group (Fig.5A). The apoptosis percentage was 4.16% in the control group (Table 1). More apoptotic cells were detected in the treatment groups (the apoptosis index was from 16.52% to 47.85%). As shown in the Table 1, the apoptosis index (AI) in B, C and D groups displayed sequentiallydecreased and dose-dependent manner, when compared with the control group(P＜0.01).

Fig.5 Apoptosis cells assayed with TUNEL methods

Table1 Effect of LBP on the apoptosis index of Hela cells (n=6)

3 Discussion

Apoptosis gives some clues to effective therapy for tumors, with many chemotherapeutic agents reportedly showing their antitumor effects by inducing apoptosis in cancer cells[24]. The report herein revealed that LBP exerts antiproliferative action and growth inhibition in cultured human cervical carcinoma Hela cells.

In this study, LBP was found to have obviously cytotoxic effect on Hela cells at the dose of 6.25 mg/L. Then we conducted the morphological study to explore whether the cytotoxic effect was related with the apoptotic process. Distinct morphological changing of apoptosis cells such as cell shrinkage, chromatin condensation and formation of apoptotic bodies were observed with AO/EB double-labeling method by fluorescence microscope. LSCM has been wildly applied to cell biology including morphological identification of apoptosis, organization of chromatin, apoptotic DNA fragmentation, endonuclease activity and the concentration of Ca2+[25]. With excited wavelength of 502 nm, the compact mass of chromatin in nuclear of cells treated with LBP was visible directly. While the excited wavelength was 460 nm, RNA could be observed in the color of red, and increasing RNA can be seen under LSCM. Transmission electron microscopy (TEM) is the most reliable method to observe the cells, apoptosis morphology. In this study we observed the typical apoptosis morphology of Hela cells: disappearance of microvilli, cell shrinkage and chromatin condensation without disruption of organelles.

As apoptosis biomarker, TUNEL is especially used to detect the programmed cell death. In this article, the nuclei with the characteristic TUNEL labeling as the typical morphological feature of apoptosis were observed in the specimens treated by LBP. The apoptosis index (AI) decreased downward from B, C and D groups with dose-dependent manner when compared with the control group (P＜0.01). The results suggested that the LBP could induce the apoptosis of the Hela cells.

In conclusion, this study showed that LBP could inhibit the growth of cancer cells and typical apoptotic morphological changes were observed by fluorescence microscope, LSCM and TEM. TUNEL assay also confirmed cells treated by LBP showed typical apoptotic features. According to these results, the LBP inhibited Hela cell proliferation through the apoptotic pathway and it is suggested that the LBP are valuable for the development of anticancer drugs.

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枸杞多糖誘導人宮頸癌Hela細胞凋亡的形態學研究

朱彩平1，張聲華2，肖軍霞2
(1.陜西師范大學食品工程與營養科學學院，陜西 西安 710062；2.華中農業大學食品科學技術學院，湖北 武漢 430070)

采用熱水法提取藥材枸杞子中的枸杞多糖。本研究通過MTT實驗發現3.125～200mg/L質量濃度范圍內的枸杞多糖能顯著抑制宮頸癌Hela細胞的增殖；采用熒光顯微鏡、透射電鏡和激光共聚焦掃描顯微鏡觀察發現經枸杞多糖處理過的Hela細胞呈現出典型的凋亡特征；原位末端脫氧核苷酸轉移酶標記法進一步證實經枸杞多糖處理過的Hela細胞呈現凋亡特征。結果提示枸杞多糖是一種潛在的抗癌復合物，其關鍵作用機制是誘導癌細胞凋亡。

枸杞多糖；人宮頸癌Hela細胞；凋亡；形態學

R151.1

A

1002-6630(2010)19-0329-06

2010-02-03

中央高校基本科研業務費專項(GK200902044)

朱彩平(1979—)，女，講師，博士，研究方向為功能食品、天然活性成分。E-mail：zcaiping@snnu.edu.cn