Experimental Study On The Inhibitory Effect Of Elemene On Laryngeal Squamous Carcinoma In Vitro And In Vivo

Experimental Study on the Inhibitory Effect of Elemene on Laryngeal Squamous Carcinoma in Vitro and in VivoIntroductionThe laryngeal squamous cell carcinoma(LSCC) is one of the familiar malignant tumors in Northern China. Along with the continuous developing of diagnosis and treatment technic, there is obviously progress in the reservation of laryngeal function, and the survival time of the patient has been longer than before. But the therapeutic efficacy of advanced stage cancer is still not as good as what we hoped. The patients with advanced stage cancer died mainly from locoregional recurrence and locoregional lymph node metastasis. Recently, it is reported that induction chemotherapy and concurrent chemoradiotherapy is more effective to control the tumor recurrence and locoregional lymph node metastasis. However, the antitumor drugs used clinically have often adverse toxicity, such as leukocytes and platelet toxicity, liver and kidney toxicity, infection and even death. Therefore, it is urgent to seek more effective anticancer drugs with low adverse toxicity.Elemene (Lanxiangxi), derived from herb (Rhizorra Zeccariae) used in Chinese medicine, has shown to exhibit the characteristic of wide spectrum of antitumor activity, improving immunofuction, synergetic effect with chemoradiotherapy, alleviating the aches caused by cancer, increasing leukocyte and so on. Most importantly, it has low adverse toxicity by contrast with the most drugs used clinically. But its antitumor mechanism on laryngeal squamous cell carcinoma is not very clear. Therefore, our study tried to investigate the inhibitory effect of Elemene on human laryngeal squamous carcinoma in vivo, and discuss its antitumor mechanismMaterials and Methods MaterialsElemene was purchased from Jingang Pharmaceutical Co.Dalian, China. The human laryngeal squamous carcinoma Hep-2 cell line was provided by the Cell Bank of Chinese Institute of Biochemistry and Cell Biology. Five-six week-old athymic nude mice (BALB/c) were obtained from Shanghai SLAC Laboratory Animal CO.LTD. The mice were kept in laminar air-flow benches. This animal study was approved by the Institutional Animal Care and Use Committee of China.Methods1,Cell culture and cytotoxicity assayLaryngeal carcinoma Hep-2 cell line was cultured in RPMI1640 with 10% fetal bovine serum in CO2 incubator for 24h, and then it was treated by elemene of 0(control),10,20,30......and 120μg/ml for 24h. The inhibiting effects of elemene on the Hep-2 cell line were evaluated by MTT Colorimetric assay. The apoptosis rate after staining with Propidium iodide (PI) and cell cycle distrabution was analysed by FCM. The morphologic evidence of apoptosis was observed by transmission electronic microscope.2,Transplantation of Hep-2 cells as solid tumors in nude mice and treatment of animals with elemeneBALB/c-nu/nu nude mice model bearing laryngeal squamous cell carcinoma was established by using human laryngeal carcinoma cell line (hep-2). Briefly, laryngeal carcinoma Hep-2 cell line was cultured in RPMI1640 with 10% fetal bovine serum in CO₂ incubator. When confluence reached 80%, cultured cells in monolayer were trypsinized and harvested by centrifugation. The Hep-2 cells (1×10⁹/L) were collected in 0.2 ml of PBS, and these cell suspensions were injected subcutaneously into the posterosuperior neck of the right side of nude mice using a 27-gauge needle. When the transplantable tumor size is more than 10mm, the mice were randomized into two group and the one group war given elemene (120 mg·kg^-1) by intraperitoneal injection (n=20) and the others PBS (n=20), at the day of 1,4,7,10,13,16,19 and 22 respectively, and meanwhile the volumes of the transplanted tumors were measured. The largest and smallest diameters were measured using a vernier caliper, and the volumes of the tumors were estimated according to the formula: V=0.52.a².b, where V is the tumor volume in mm³, and a and b are the smallest and largest tumor diameters in millimeter, respectively. The animals were executed by cervical dislocation three days after the last treatment, and full necropsies were performed. Tumors were dissected and weighed individually. The percent inhibition rate (IR) was calculated: IR = (1-Tumor weight test/Tumor weight control). The mean tumor volume,tumor weight,mice net weight and tumor inhibition rate were evaluated.Part of the tumor tissue was fixed in 10% formalin and embedded with paraffin for HE staining and immunohistostaining, and the other part was kept immediately in refrigeratory at -80℃for RT-PCR assay.3,Immunohistochemistry for the expressions of Ki67, VEGF-C and VEGFR-3 proteinImmunohistostaining was performed on 4μm sections of formalin-fixed and paraffin-embedded tumor tissues using streptavidin peroxidase (SP) method. The SP-900 (Histosain^TM-plus kits) and the primary antibody purchased from Beijing Zhongshan goldenbridge biotechnology co.LMD. The primary antibodies include polyclonal rabbit anti-human against VEGF-C (ZA-0266), polyclonal rabbit anti-human against VEGFR-3(ZA-0267) and rabbit anti-Ki-67 nuclear antigen monoclonal antibody (ZA-0502). The procedures were followed by the manufacture's instruction and the tissues were counter-stained with hematoxylin. The negative control was prepared in the PBS and the positive control for staining was obtained from the company. The positive were defined as brown to yellow staining in nucleolus for Ki67 and in cytoplasm for VEGF-C and VEGFR-3. The positive percentage was adopted to describe the results of Ki67 and the semiquantitative integration was adopted to describe the results of VEGF-C proteins. The VEGFR-3 positive vessel density was calculated by Weidner's method.4,RT-PCR assay for VEGF-C and VEGFR-3 mRNA expressionAccording to manufacture's instructions, total RNA from each sample (50-100mg tumor tissue) were isolated by using Tripure Isolation Reagent (Roche Applied Science) and the concentration and purity of RNA were detected by DNA/RNA detector. The reverse transcription solution consists of 25mMMgSO₄ 4μl, 2×RNA PCR Buffer 10μl, RNase Free dH₂O 0.5μl, 10mMdNTP 1μl, RNase inhibitor 0.5μl, AMV reverse tanscriptase 1μl, Oligo dT-Adaptor Primer 1μl and RNA 2μg. The reverse transcription reaction were performed by the conditions as follows: 1 min at 65℃, 30min at 30℃, 15-30min to 65℃3min, 5min at 98℃, 5min at 50℃.According to the reference, VEGF-C, VEGFR-3 and GAPDH (Glyceraldehyde phosphate dehydrogenase) specific primers were synthesized by Invitrogen Biotechnology Co.. Sequences of specific primer for reverse transcription-polymerase chain reaction (RT-PCR) were as follows: VEGF-C (592bp); sense: 5'-AGTTTTGCC AATTCACACTTCCTG-3', antisense: 5'-GTCATTGGCAGAAAACCAGTCTT-3' VEGFR-3(298bp); sense: 5'-AGCCATTCATCAACAAGC CT-3', antisense: 5'-GGCAACAGCTGGATGTCATA-3'. GAPDH(188bp); sense: 5'-AATCCATGGC ACC GTCAA GGCT-3', antisense: 5'-TCAGGTCCACCACTG ACACGTT-3'.PCR amplification was performed on a PCR thermal cycler (Thermo EC, Micramax RF, USA). 3μl cDNA mixture was subject to amplification in solution containing 10×PCR buffer 2.5μl, 10mM dNTP 2μl, sterile purified water 16.7μl, Taq DNA polymerase 0.2μl, sense primers 0.5μl and antisense primers 0.1μl. PCR conditions were initially porformed by denaturation for 3min at 94℃, followed by 30 cycles of denaturation of 94℃for 1min and annealed at 55℃for 1min30sec, finally by extension at 72℃for 2min. The efficiency was detected by GAPDH. PCR products were separated on a 1.5% agarose gel and the bands were visualized by ethidium bromide staining, then ascertained with the comparison to DL2000 DNA Markers (TaKaRa Co.). Band Leader Ver. 3.0 software was used to analysize electrophoresis strip and the relative values were obtained by calculating the ratio of VEGF-C/GAPDH and VEGFR-3/GAPDH.5,Statistical analysisChi-squared test was used in data of quantity. T-rest or ONE-WAY ANOVA method was used in data of quality. Spearman method was applied for the correlation analysis.The data were analyzed by the SPSS for windows 13.0.Results1,Cytotoxicity assayHep-2 cell growth was significantly inhibited by elemene in vitro and the IC₅₀ of elemene on Hep-2 cell was 62.70μg/ml at 24h. The apoptosis cells increased with the increase of drug concentration and the elapse of time. The cell cycle of Hep-2 cells were arrested by elemene in the G₂/M phase.Chromatin concentration and crescent form along nuclei membrane were observed under transmission electron microscopy.2,Treament with elemeneElemene could inhibit the tumor growth in vivo without definite side effect. The differences were statistically significant for the mice net weight, tumor weight, and tumor volume between the treatment group and the control group. The tumor inhibition percentage was 52.24.3,ImmunohistochemistryKi67 index was 28.88±3.15 in elemene treatment group and 78.18±4.51 in control group, respectively; and the t test showed that P＜0.01 and the differences were statistically significant. The grade of VEGF-C protein expression was 15.82±2.05 in treatment group and 113.61±16.29 in control group, respectively; and the t test showed that P＜0.01 and the differences were statistically significant. The VEGFR-3 positive vessel density was 7.02±0.59 in treatment group and 19.84±1.89 in control group, respectively; and the t test showed that P＜0.01 and the differences were statistically significant.4,RT-PCR assay:The gene expression of VEGF-C and VEGFR-3 of the treatment group is lower than that of the control group, and the t test showed that P＜0.01 and the differences were statistically significant.Conclusions1,In vitro:Elemene could significantly inhibit the growth of human laryngeal squamous carcinoma.Elemene could induce apoptosis on Hep-2 cell and arrest the cell cycle in G₂/M phase.2,In vivo: (1) Elemene could significantly inhibit the growth of human laryngeal squamous carcinoma without definite side effect.(2) Elemene could inhibit the expression of Ki67 of transplantable carcinoma in nude mice. This may be one of the antimumor mechanisms of elemene.(3) Elemene could inhibit the expression of VEGF-C and VEGFR-3 and decrease the VEGFR-3 positive vessel density of transplantable carcinoma in nude mice.This may be another important antimumor mechanism of elemene.