The fusiform skin paddle in fibula free flap: a fusiform-designed skin paddle for maxillofacial soft defect reconstruction and reducing leg wound tension.

Objective: To investigate the feasibility of leg wound closure and reconstruction of maxillofacial soft defect by a fusiform-designed skin paddle in fibula free flap (FFF). Methods: Fifty patients who underwent FFF for reconstruction of maxillofacial soft defect were divided into two groups. The fusiform group (20 patients) was treated using a fusiform-designed skin paddle in FFF (skin paddle width less than 2 cm), and leg wound was closed using primary suturing. Reconstruction of the maxillofacial soft defect or filling of dead space was achieved by folding the fusiform skin paddle. The conventional group (30 patients) was treated using the conventional-designed skin paddle (skin paddle width no less than 2.5 cm). The leg wound was closed using mattress suturing or skin graft, while reconstruction of the maxillofacial soft defect or filling of dead space by conventional way. The average postoperative length of hospital stay, healing time of leg wound, and post-surgical complications were recorded at least 6 months after the surgery. Results: Compared with traditional method, the fusiform-designed skin paddle reduced the average healing time of the leg wound (fusiform group: 11.05 days, conventional group: 14.77 days, P < 0.05). The average length-to-width ratio in fusiform group was significantly greater than that of in conventional group (fusiform group: 5.85, conventional group: 2.93, P < 0.05), and no difference was observed on the graft size of skin paddle between two groups (fusiform group: 23.13, conventional group: 27.13, P > 0.05). The post-surgical early complications of the leg wound in the conventional group were higher than that of in the fusiform group (fusiform group: 0%, conventional group: 6.67%), while the post-surgical late complication of the donor site between the two groups showed no case. Healing disorders of maxillofacial soft reconstruction in the conventional group were higher than that of in the fusiform group (fusiform group: 5.26%, conventional group: 20.69%). Conclusions: Fusiform-designed skin paddle for closure of the leg wound and maxillofacial soft defect is a feasible alternative to the conventional- designed skin paddle. The fusiform- designed skin paddle resulted in the less postoperative length of hospital stay, shorter healing time of leg wound and less complication.

Retinoic acid inhibits osteogenic differentiation of mouse embryonic palate mesenchymal cells.

BACKGROUND: All-trans-retinoic acid (ATRA), a known teratogenic factor affecting the development of cleft palate, has been shown to adversely affect craniofacial development. In the present study, we evaluated the effects of ATRA on the osteo-/adipogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells, which served as a valid model system for investigating the mechanisms regulating osteogenesis during palatogenesis. METHODS: MEPM cells were derived from gestational day 13 C57BL/6N mouse embryos and induced to differentiate in the presence or absence of ATRA in either osteogenic medium (OM) or control medium (CM). RESULTS: Alkaline phosphatase (ALP) activity assays, von Kossa staining, and RT-PCR assays confirmed that MEPM cells underwent osteogenic differentiation when cultured in OM. Although ATRA induced ALP activity and lipid accumulation in MEPM cells, it failed to induce matrix mineralization and osteoblastic gene expression. BMPR-IB and Smad5 mRNA levels increased significantly in cells cultured in OM and declined following treatment with ATRA, whereas the expression of the BMPR-IA mRNA was up-regulated by ATRA. CONCLUSIONS: In conclusion, our results suggested that ATRA and the BMP signaling pathway cooperate to inhibit osteogenesis and promote adipogenesis of MEPM cells.

Survivin shRNA induces caspase-3-dependent apoptosis and enhances cisplatin sensitivity in squamous cell carcinoma of the tongue.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family; it is overexpressed in most cancer tissues and induces resistance to chemotherapy. In this study, we investigated whether a short hairpin RNA (shRNA) targeting survivin can induce apoptosis and enhance chemosensitivity to cisplatin in squamous cell carcinoma of the tongue. Results showed that chemosensitivity to cisplatin was surviving dependent in three cell lines (Tca8113, Bca885, and MCF7); higher survivin mRNA expression levels were associated with lower sensitivity to cisplatin. A plasmid-containing survivin shRNA was constructed and transfected into cell line Tca8113. Survivin shRNA inhibited expression of survivin mRNA and protein (63% and 65% inhibition, respectively), significantly inhibited cell proliferation, and enhanced chemosensitivity to cisplatin (p < 0.05). Apoptosis and caspase-3 activity were induced when cells were treated with survivin shRNA and/or cisplatin. Survivin shRNA induced caspase-3-dependent apoptosis and enhanced chemosensitivity to cisplatin in these tongue squamous cell carcinoma cell lines.

Antitumor effects of hydroxycamptothecin-loaded poly[ethylene glycol]-poly [gamma-benzyl-L-glutamate] micelles against oral squamous cell carcinoma.

Therapeutic use of hydroxycamptothecin (HCPT), a promising antitumor agent, is limited by its poor solubility and rapid destruction. Amphiphilic block copolymer micelle carriers possess significant potential for improving drug solubility and stability. Poly[ethylene glycol]-poly[gamma-benzyl-L-glutamate] (PEG-PBLG) micelles were prepared and loaded with the active lactone form of HCPT using an uncomplicated dialysis method. HPLC and scanning electron microscopy studies revealed an encapsulation efficiency of 56.8% and a core-shell figure with a mean diameter of 200 nm. Encapsulated HCPT lactone was compared with the less active, open ring-carboxylated HCPT-Na+ soluble form generated in vivo from the free active lactone for activity against oral squamous cell carcinoma. Cytotoxicity in vitro was measured in cultured Tca8113 cells by the MTT assay and microscopy techniques. The golden hamster cheek pouch squamous cell carcinoma model was employed for in vivo studies; encapsulated lactone and open ring-carboxylated forms of HCPT were administered intraperitoneally, followed by determinations of tumor growth rate and inhibition ratio. PEG-PBLG micelles were not cytotoxic in vitro. At 48 h of treatment, open ring-carboxylated HCPT proved significantly more cytotoxic in vitro than encapsulated HCPT lactone. At 96 h, however, the open ring-carboxylated and encapsulated drugs displayed comparable in vitro cytotoxicities. In the in vivo squamous cell carcinoma model, encapsulated HCPT lactone produced greater and more prolonged tumor suppression compared to the open ring-carboxylated form. The antitumor effects of HCPT/PEG-PBLG micelles against oral squamous cell carcinoma in vivo are concluded to be superior to those exerted by open ring-carboxylated HCPT.

In vitro and in vivo effect of 5-FC combined gene therapy with TNF-α and CD suicide gene on human laryngeal carcinoma cell line Hep-2.

This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, (1)Hep-2/TIC group; (2)Hep-2/CD group; (3)Hep-2/TNF-α group; (4)Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.

[Synthetic radiation-inducible promoter mediated CDglyTK gene in treatment of Tca8113 cells].

OBJECTIVE: To observe the therapeutic effect of CDglyTK gene mediated by synthetic radiation-inducible promoter in the treatment of Tca8113 cells. METHODS: CDglyTK gene in pCEA-CDglyTK was subcloned into pcDNA3.1 (+) to construct plasmid pcDNA3.1 (+)-CDglyTK, and then the synthetic radiation-inducible promoter in pMD18 -T -E was inserted into pcDNA3.1 (+) -CDglyTK to construct plasmid pcDNA3.1 (+ )/E -CDglyTK. The recombinant plasmid was transfected into Tca8113 cells by lipofectamine, and then exposed to 3 Gy irradiation. Cytotoxicity was evaluated by MTT. The expression of CDglyTK gene was detected by RT-PCR. The apoptosis and proliferation were examined by flow cytomtery. RESULTS: The plasmid pcDNA3.1 (+)/E-CDglyTK was constructed successfully. The comparative survival rate of Tca8113 cells was markedly decreased by induction irradiation. Up-regulation of CDglyTK expression was found in Tca8113 cells exposed to irradiation. The apoptosis index (AI) of Tca8113 cells exposed to irradiation was higher than that of Tca8113 cells without irradiation, the other way round, the proliferation index (PI) of Tca8113 cells exposed to irradiation was lower than that of Tca8113 cells without irradiation. CONCLUSION: The synthetic radiation-inducible promoter can be served as a molecular switch to improve the expression of CDglyTK gene in Tca8113 cells, and low dose induction radiation can significantly improve the therapeutic efficiency.

[Radiation-inducible promoters-mediated cdglytk gene in the treatment of buccal carcinoma in golden hamster].

OBJECTIVE: To observe the therapeutic effect of CDglyTK gene mediated by radiation-inducible promoters in the treatment of buccal carcinoma in Golden Hamster. METHODS: Animal models of buccal carcinoma in golden hamster were established by painting 0.5% dimethyl-benzanthracene. The plasmids pcDNA (+) 3.1/E-CDglyTK were transfected into tumors by lipofectamine. 24 h later, the tumors were exposed to 3 Gy irradiation. Animals were monitored at regular intervals for volume of tumors. CDglyTK mRNA was assayed by RT-PCR. Apoptosis and proliferating cell nuclear antigen were detected respectively by in situ end-labeling and immunohistochemical methods. RESULTS: Compared with control groups, the tumor was suppressed obviously by CDglyTK gene therapy combined with 3 Gy induction radiation. The expression of CDglyTK gene could be detected by RT-PCR in the transfected tumor, and up-regulation of CDglyTK expression was found in tumor exposed to radiation (P < 0.05). There was significant difference in apoptosis index or proliferation index between tumor without irradiation and tumor with irradiation (P < 0.05). CONCLUSIONS: The radiation-inducible promoter can be served as a molecular switch to regulate the expression of CDglyTK gene in buccal carcinoma in golden hamster, and low dose induction radiation can significantly improve the therapeutic effects.

[Antitumor effects of ring-closed and ring-opened hydroxycamptothecin on oral squamous carcinoma cell line Tca8113].

BACKGROUND & OBJECTIVE: The antitumor effect of hydroxycamptothecin (HCPT) closely relates with its lactone form (including ring-closed form and ring-opened form), but the antitumor effects of ring-closed HCPT (C-HCPT) and ring-opened HCPT (O-HCPT) remain controversial. Researches have showed that O-HCPT has obvious in vitro and in vivo antitumor effects on oral squamous cell carcinoma. This study was to compare the antitumor effects of C-HCPT and O-HCPT on oral squamous carcinoma cell line Tca8113, and explore the mechanisms. METHODS: Tca8113 cells and its xenografts in BALB/C nude mice were treated with C-HCPT and O-HCPT. The cytotoxicity of HCPT was measured by MTT assay. Cell cycle was detected by flow cytometry. The growth state of Tca8113 cells xenografts was observedû the tumor doubling time and inhibition rate were calculated. The concentration of total HCPT in plasma and tumor tissue was quantitated by high-performance liquid chromatography (HPLC); the pharmacokinetic parameters were estimated. RESULTS: C-HCPT and O-HCPT showed similar cytotoxicity effects on Tca8113 cells in vitro. Low concentration of HCPT (< 1 micromol/L) arrested cell cycle of Tca8113 cells at S phase and G(2)/M phase; high concentration of HCPT (100 micromol/L) obviously induced apoptosis of Tca8113 cells. Compared with control group, the xenografts of HCPT-treated group grew slowly, and the tumor doubling time was prolonged. The tumor inhibition rates were 69.6% (3 mg/kg of O-HCPT), 65.0% (3 mg/kg of C-HCPT), and 74.1% (10 mg/kg of O-HCPT), respectively. The plasma AUC was 2.66 microg . h . ml(-1) for C-HCPT (10 mg/kg) and 0.42 microg . h . ml(-1) for O-HCPT (10 mg/kg). HCPT could be detected in tumor tissue. No obvious toxicity was observed in 3 mg/kg of HCPT group; obvious gastrointestinal reaction was observed in 10 mg/kg of O-HCPT group; all nude mice in 10 mg/kg of C-HCPT group died 2 days after treatment. CONCLUSIONS: Both C-HCPT and O-HCPT have strong in vitro and in vivo cytotoxic effects on Tca8113 cells, which relate to cell cycle arrest and cell apoptosis inducement. Cytotoxicity of C-HCPT is more severe than that of O-HCPT.

[All-trans retinoic acid augments the bystander effect of herpes simplex virus thymidine kinase/ganciclovir system in the treatment of tongue carcinoma cell line].

OBJECTIVE: To investigate the augmentation effect and mechanism of all-trans retinoic acid (ATRA) on the bystander effect of herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system in the treatment of tongue carcinoma cell line. METHODS: Immunocytochemistry and flow cytometry (FCM) were used to analyze the expression of Cx43 protein in Tca8113 cells after treated with ATRA; The bystander effect of HSV-TK/GCV system on tongue carcinoma cells before and after treatment with ATRA was detected by MTT assays. The interaction of ATRA and bystander effect was analyzed by factorial experiment. RESULTS: After treated by ATRA (10(-7) mol/L - 10(-5) mol/L), the expression of Cx43 protein was up-regulated in Tca8113 cells and the positive rate of Cx43 protein increased from 5.17% (before treatment) to 30.53% (10(-5) mol/L ATRA). There was significant difference between ATRA treated cells and untreated cells (P < 0.01). The bystander effect of HSV-TK/GCV system on Tca8113 cells was poor, but improved after combined with ATRA. There was cooperation effect between ATRA and bystander effect of HSV-TK/GCV (P < 0.05). CONCLUSIONS: ATRA can augment the bystander effect of HSV-TK/GCV system in the treatment of tongue carcinoma cell line. The mechanism might be due to up-regulation of Cx43 gene and restore gap junction intercellular communication.

[Studies on 5-FU/PEG-PBLG nano-micelles: preparation, characteristics, and drug releasing in vivo].

BACKGROUND & OBJECTIVE: 5-Fluorouracil (5-FU) belongs to antimetabolic anticancer drugs and its half-life time in vivo is only about 5 minutes. Continuous infusion (48 hours,72 hours,28 days) was commonly used to maintain long-time steady-state concentration (Css). So it is necessary to exploit new slow-release system for 5-FU. This article was designed to investigate the preparation technique,shape characteristics, and drug releasing characteristics in vivo of 5-FU loaded core-shell type nanoparticles which were made by biodegradable amphiphlic poly (ethylene glycol)-poly (gamma-benzyl-L-glutamate)(PEG-PBLG). METHODS: The PEG-PBLG nano-micelles were prepared by diafiltration method. Its morphology was observed by transmission electron microscopy and scan electron microscopy. Encapsulating efficiency of 5-FU was determined by ultraviolet spectrophotometry. The 5-FU releasing characteristics from nano-micelles in vivo were investigated by high-performance liquid chromatography (HPLC). RESULTS: 5-FU loaded PEG-PBLG copolymer nano-micelles (5-FU/PEG-PBLG) was of round or ellipsic shape. The diameter of the micelles was 180-250 nm; the size of drug storeroom was about 200 nm and the thickness of hydrophilic zone was about 30 nm. The entrapment efficiency was (29.5+/-0.015)%. In control group, 5-FU is one-compartment in vivo; its half-life was 5.3 minutes; the maximum plasma concentration (C(max)) was 17047.3 microg/L; T(max) was the time of attaining C(max) of the end of infusion; the area under the plasma concentration-time curve (AUC) was 6263.7 microg/L x min. However, the model of nano-micelles in vivo was typical release-drug of nano-micelles namely abrupt release-slow release; its t(1/2) was 63.2 minutes; C(max) was 4563.5 microg/L; T(max) was 1.25 hours; AUC was 5794.5 microg/Lxmin. Compared with 5-FU preparation, T(max) of 5-FU/PEG-PBLG was longer (P< 0.01);C(max) was lower (P< 0.01);t(1/2) was longer (P< 0.01); AUC was similar (P >0.01). CONCLUSION: The 5-FU/PEG-PBLG nano-micelles can change the pharmacokinetics of 5-FU, prolonging the circulation time in vivo and make a slow release.

[Anticancer effect of hydroxycampothecin on oral squamous carcinoma cell line].

BACKGROUND & OBJECTIVE: Studies have demonstrated that hydroxycamptothecin (HCPT) had therapeutic effect on many malignant tumors, but few studies on oral squamous cell carcinoma were reported. This study was designed to investigate the anticancer effect and mechanism of hydroxycampothecin (HCPT) on oral squamous carcinoma cell line (Tca8113). METHODS: The cytotoxicity of HCPT on Tca8113 cell line was measured by MTT assay and cell cycle detected by flow cytometry(FCM). The growth status of Tca8113 cell xenografts following treatment with HCPT was observed. The doubling times and tumor inhibition rate were calculated. RESULTS: HCPT had strong cytotoxicity on Tca8113 cells, the IC50 was 2 mumol/L. After treatment with low concentration of HCPT, the cell cycle was arrested in S phase or G2 + M phase; while at high concentration, apoptosis was obviously found and the rate of apoptosis was increased as the time and concentration of HCPT. Compared with control group, the xenografts of HCPT treated group grew slower and tumor doubling time was prolonged, The tumor volumes of HCPT treated group at 28th day were significantly smaller(P < 0.001) with tumor inhibition rate of 69.6%. CONCLUSION: The result showed that HCPT had strong cytotoxicity effect to oral squamous carcinoma cells and significant growth inhibition on Tca8113 xenografts. HCPT can arrest cell cycle in S phase and G2 + M phase and induce cell apoptosis.

[Meta analysis of the relationship between tumorigenesis of oral squamous cell carcinoma and human papillomavirus infection].

BACKGROUND & OBJECTIVE: Previous researches showed that human papillomavirus (HPV) infection was closely related to tumorigenesis of oral squamous cell carcinoma (OSCC). But the results of these studies have great differences because of different research methods. This study was to evaluate synthetically the relationship between OSCC of Chinese and HPV infection by meta analysis. METHODS: From Jan. 1990 to Apr. 2003, 44 references reported about the relationship between tumorigenesis of OSCC of Chinese and HPV infection were collected from Chinese Biomedical Literature Analysis and Retrieval System for Compact Disc (CBMdisc). There were 10 references accorded with research criteria which was case-control study, and detected by polymerase chain reaction (PCR). Fisher and meta analysis were used to quantitatively and qualitatively analyze these references synthetically. RESULTS: The synthetic qualitative analysis by Fisher showed there was significant association between the tumorigenesis of OSCC and HPV infection (P< 0.005). The synthetic quantitative analysis by meta analysis revealed that the combined odds ratioes (ORc) for HPV, and HPV16 infection of OSCC were 8.89 (3.62-21.80), and 6.81 (2.18-21.32) times that of normal oral mucosa. The mean positive rates of HPV, and HPV16 detection in OSCC were 49.02% (36.48%-61.57%),and 45.74% (31.94%-59.54%) higher than that of normal oral mucosa. CONCLUSION: Human papillomavirus infection, especially HPV16, will increase the risk of tumorigenesis of OSCC.