[Effect of standardized treatment on reactivity of toe microcirculation in patients with type 2 diabetes mellitus].

OBJECTIVE: To evaluate the effect of standardized treatment on reactivity of the lower limb terminal microcirculation in patients with type 2 diabetes mellitus (T2DM) by high frequency ultrasound combined with warm bath. METHODS: A total of 66 patients with T2DM were collected from June 2014 to May 2015 in the Second Affiliated Hospital of Wenzhou Medical University.According to the vascular complications , the patients were divided into group A without complications (36 cases)and group B with complications (30 cases). Hemodynamic parameters such as peak systolic velocity(PSV), end-diastolic velocity(EDV) and resistance index(RI) of the right plantar digital artery on fibular side of the 1st toe were acquired through Doppler in all subjects.Then the above operation were performed repetitively on all subjects after the right foot was immersed in 40 ℃ warm water for 5 minutes.The change rates of PSV, EDV and RI were calculated after warm bath.All subjects were examined again after three months treatment .The growth rates of the change rate of PSV, EDV and RI were calculated after treatment.All parameters were analyzed. RESULTS: The change rates of the parameters in group B before and after treatment were lower than those of group A, before treatment (0.108±0.077 vs 0.184±0.091, P=0.037, 0.184±0.101 vs 0.380±0.167, P=0.002, 0.007±0.004 vs 0.015±0.008, P=0.028 7), after treatment (0.155±0.111 vs 0.421±0.138, 0.287±0.108 vs 0.794±0.286, 0.012±0.008 vs 0.039±0.014, P=0.000); the post treatment growth rates of the change rates of the parameters in group B were all less than those of group A (0.414±0.303 vs 2.192±2.673, P=0.048, 0.660±0.406 vs 1.422±1.075, P=0.042, 0.633±0.830 vs 2.191±2.269, P=0.048). The change rates of the parameters in two groups after treatment were higher than those before treatment (all P<0.05). CONCLUSION: Warm bath test can be used to detect change degree of hemodynamic parameters in patients with T2DM so as to evaluate reactivity of blood microcirculation, which has a certain clinical application value in treatment.

Marsdenia tenacissima extract sensitizes MG63 cells to doxorubicin-induced apoptosis.

Marsdenia tenacissima extract (MTE) is a new plate-derived biotechnology product that is frequently used, but occasionally reported, in the field of chemotherapy. In this study, we assessed the antitumor activity and related mechanisms of MTE by various biotechnological methods. The survival rates of MG63 osteosarcoma cells treated with MTE and doxorubicin were measured, individually or jointly, and the changes in cellular shape, apoptotic rates, and Fas expression were observed. The results indicated that combination of MTE and doxorubicin up-regulated Fas expression and induced apoptosis. The survival rate of combined application of 50 mg/mL MTE and 1 µg/mL doxorubicin was significantly lower than that of the individual application (P < 0.01). Other biotechnology methods also showed an apoptosis-inducing effect of combined application that was much stronger than individual application. All of these results suggested that MTE may promote the effects of doxorubicin chemotherapy, perhaps related to the up-regulation of Fas expression in tumor cells.

Synergistic increase in the sensitivity of osteosarcoma cells to thermochemotherapy with combination of paclitaxel and etoposide.

Osteosarcoma is a malignant bone tumor which is found most commonly in adolescents and young adults. Local perfusion thermochemotherapy has long been proposed as an alternative strategy for the treatment of osteosarcoma. As a standard anticancer drug, paclitaxel plays a significant role in the treatment of a number of tumors; however, little is known concerning its ability to promote thermochemotherapy. The aim of this study was to evaluate the cytotoxic effects of a combination of paclitaxel and etoposide on an osteosarcoma cell line in the presence of hyperthermia and to investigate the related mechanism. Our study indicated that 1 h after the application of a combination of 10 µg/ml paclitaxel and 5 µg/ml etoposide to OS732 cells at 43˚C, the survival rate of the cells was 14.52% which was significantly lower than when either 10 µg/ml paclitaxel (45.83%) or 5 µg/ml etoposide (43.31%) was applied alone (P<0.01). Moreover, changes in cellular morphology and apoptotic rates indicated that the apoptosis-inducing effect of the combination was much stronger than that of either drug applied individually. Fas expression levels in the OS732 cells were increased by the combination of paclitaxel and etoposide in the presence of hyperthermia. Therefore, paclitaxel enhances the thermochemotherapy of the osteosarcoma cell line and this is primarily accomplished by the upregulation of Fas expression and the induction of apoptosis.

Differential effects of chromosome 3p deletion on the expression of the putative tumor suppressor RAR beta and on retinoid resistance in human squamous carcinoma cells.

Retinoids' effects on cell growth and differentiation are mediated by nuclear retinoid receptors, which are ligand-activated transcription enhancing factors. Because the expression of the retinoic acid receptor beta (RARbeta) gene, which is located on chromosome 3p24, is diminished in premalignant and malignant tissues it has been proposed that it acts as a tumor suppressor. To test the hypothesis that RARbeta loss leads to retinoid resistance, we studied several karyotyped head and neck squamous carcinoma (HNSCC) cell lines (UMSCC-17A, -17B, -22A, -22B, and -38) with deletion of one chromosome 3p arm. RARbeta mRNA was neither detected nor induced by retinoic acid in these cells, whereas it was expressed and induced by retinoic acid in two other HNSCC cell lines (1483 and 183) without 3p deletion. Methylation of the RARbeta gene promoter was detected in the 17B and 22B cells that failed to express RARbeta but no methylation was found in 183A cells that did express RARbeta mRNA. Responsiveness of HNSCC cells to several retinoids in assays of growth inhibition and colony formation, was rank ordered as: 22B>1483>38>183>17B. Additionally, retinoid response elements were transactivated in 22B more efficiently than in 17B cells. These results indicate that loss of RARbeta expression does not necessarily lead to loss of growth inhibition by retinoids or to a block of retinoid signaling.

Effects of sodium butyrate on growth, differentiation, and apoptosis in head and neck squamous carcinoma cell lines.

BACKGROUND: Biologic agents that reverse early changes in the aerodigestive tract mucosa have potential treatment applications for patients with field cancerization of the upper aerodigestive tract. Sodium butyrate (BA) is a normal dietary constituent that induces differentiation and inhibits growth in several malignant cell types in vitro, but its effect on head and neck squamous cell carcinoma (HNSCC) has not been evaluated. METHODS: Using five HNSCC cell lines, the effects of BA on cell proliferation and apoptosis were examined by colorimetric and fluorescence-labeling methods, and the expression of differentiation markers and apoptosis-related proteins were analyzed using Western and Northern blotting, flow cytometry, and cell cycle analysis. RESULTS: BA-induced growth inhibition and apoptosis in HNSCC cells at millimolar concentrations. Apoptosis induction did not depend on the p53 status of the cell lines or on expression of members of the Bcl-2/Bax family. CONCLUSIONS: These results demonstrate that butyrate has activity against HNSCC in vitro and may have clinical applications for management of HNSCC patients.

Expression of retinoic acid receptor beta is associated with inhibition of keratinization in human head and neck squamous carcinoma cells.

The preventive effects of retinoids on oral carcinogenesis may be related to their ability to modulate the growth and differentiation of human oral squamous epithelial cells. Nuclear retinoid receptors (RAR alpha, beta, and gamma, and RXR alpha, beta, and gamma) may mediate these effects by regulating gene transcription. The removal of serum from the growth medium of two head and neck squamous cell carcinoma lines 1483 and SqCC/Y1 resulted in a decrease in RAR beta mRNA level and concurrent increases in the expression of the keratin K1 and transglutaminase type I (TGase I), which are markers of differentiation of keratinizing squamous epithelial cells. All-trans-retinoic acid (tRA) or 13-cis-RA increased RAR beta and decreased K1 and TGase I mRNA levels in serum-free medium. Transcriptional activation of reporter genes by means of retinoid response elements (RARE and RXRE) indicated that the RXR-RAR pathway predominates over the RXR homodimer pathway in the 1483 cells. Among several synthetic retinoids with preference for binding to specific nuclear retinoid receptors, those that induced RAR beta also suppressed K1. The inverse association between RAR beta expression and K1 and TGase I levels implicates this receptor in suppression of keratinization in oral epithelial cells.

Higher potency of N-(4-hydroxyphenyl)retinamide than all-trans-retinoic acid in induction of apoptosis in non-small cell lung cancer cell lines.

Most human non-small cell lung cancer (NSCLC) cell lines are refractory to all-trans-retinoic acid (ATRA). Recently, N-(4-hydroxyphenyl)retinamide (4HPR) was found to induce apoptosis in various tumor cells. In this study, we compared and contrasted the effects of 4HPR and ATRA on the growth and apoptosis of 10 NSCLC cell lines and normal human bronchial epithelial (NHBE) cells. All of the cancer cell lines and the NHBE cells were sensitive to 10 microM 4HPR, and their numbers decreased to <20% of the controls after a 5-day treatment, whereas ATRA decreased cell numbers to about 50% of the controls in three cell lines and was less effective in the rest of the tumor cell lines. ATRA inhibited the growth of the NHBE cells by 70-80%. 4HPR induced apoptosis in most of the cells, including the ATRA-resistant ones, as evidenced by a DNA fragmentation assay. No correlation was found between growth inhibition by 4HPR and the expression of retinoic acid receptor beta (determined by Northern blotting and PCR), p53, or Bcl-2 proteins (analyzed by Western blotting). These results demonstrate that 4HPR is more potent than ATRA in inducing apoptosis in NSCLC cells and suggest that further clinical trials for prevention and therapy of NSCLC using 4HPR are warranted.

Retinoid refractoriness occurs during lung carcinogenesis despite functional retinoid receptors.

Retinoids have demonstrated activity in the prevention of second primary tumors in patients with non-small cell lung cancer (NSCLC). They also contribute to the normal growth and differentiation of human bronchial epithelial (HBE) cells. Because retinoids mediate their actions through retinoid nuclear receptors (RARs and RXRs), aberrant signaling through retinoid receptors could contribute to lung carcinogenesis. Using a lung carcinogenesis model consisting of normal, premalignant, and malignant HBE cells, we examined all-trans retinoic acid (t-RA)-induced changes in cellular growth. These studies revealed that t-RA treatment inhibited the growth of normal HBE cells, but premalignant and malignant HBE cells were relatively resistant to t-RA. Coincident with the development of retinoid refractoriness, basal expression of the retinoic acid nuclear receptor beta (RAR-beta) increased. Analysis of receptor function by gel shift and transient transfection assays of normal, premalignant, and malignant HBE cells demonstrated that receptor-DNA binding and transcriptional activation properties were intact in the t-RA-refractory malignant HBE cells. To compare these findings to NSCLCs in patients, we investigated retinoid receptor expression in NSCLC biopsies. A subset of the tumors expressed RAR-beta, reflecting the RAR-beta expression observed in the malignant HBE cells in culture. These findings demonstrate that retinoid receptor function was intact in the t-RA-refractory malignant HBE cell line, suggesting that the defect in retinoid signaling in this lung carcinogenesis model is not intrinsic to the retinoid receptors.

Enhanced efficacy of combinations of retinoic acid- and retinoid X receptor-selective retinoids and alpha-interferon in inhibition of cervical carcinoma cell proliferation.

Retinoic acid receptors and retinoid X receptors form heterodimers, bind to retinoic acid response elements, and transactivate the transcription of retinoid-responsive genes. Two synthetic retinoids [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid (TTAB) and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthale n ecarboxylic acid (TTNN)], which preferentially bind retinoic acid receptors, inhibited the proliferation of cervical carcinoma ME180 cells by 50% at 0.2 nM and 0.2 microM, respectively. In contrast, two other retinoids [2-(4-carboxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-1,3-dithiane (SR11203) and 4-(2-methyl-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)propenyl)benzoic acid (SR11217)], which preferentially bind retinoic X receptors, inhibited growth by only 12 and 18% at 1 microM, respectively. The combination of suboptimal concentrations of TTAB (0.1 nM) or TTNN (10 nM) with each of the retinoic X receptor-selective retinoids at 1 microM showed more than additive effects on cell proliferation, especially with SR11217. Further increases in proliferation inhibition were observed when IFN-alpha (100 units/ml) was added to these retinoid combinations. Activation of transcription of a reporter gene linked 3' to the retinoic acid receptor beta retinoic acid response element in transiently transfected cells also exhibited additive effects when the cells were treated with combinations of TTAB or TTNN with SR11217. This additive activation of transcription may be the reason why the combination of retinoids is more effective than each retinoid alone. The results also suggest that the use of combinations of retinoids and IFN-alpha may lead to enhanced antitumor effects.

Modulation by retinoic acid (RA) of squamous cell differentiation, cellular RA-binding proteins, and nuclear RA receptors in human head and neck squamous cell carcinoma cell lines.

The ability of all-trans-retinoic acid (RA) to modulate the growth and squamous differentiation of four head and neck squamous cell carcinoma cell lines (183, 886, 1483, and SqCC/Y1) was examined, and the relationship of their state of squamous differentiation and RA responsiveness to the expression of cytosolic RA-binding proteins (CRABPs), nuclear RA receptors (RARs), and retinoid X receptors (RXRs) was investigated. RA inhibited proliferation of all but the 183 cell line and suppressed squamous differentiation markers K1 keratin, type 1 transglutaminase, and involucrin mRNAs and proteins to varying degrees in 183, 1483, and SqCC/Y1 cells. Traces of CRABP-I mRNA were detected only in the 886 cells, whereas CRABP-II mRNA was detected in the other three cell lines. RA suppressed CRABP-II expression in SqCC/Y1 cells but had no effect on its expression in the other cell lines. All cell lines expressed mRNAs for RAR-alpha, RAR-beta, RAR-gamma, and RXR-alpha. The RAR-beta mRNA level was lowest in the SqCC/Y1 cells, and RXR-beta and RXR-gamma were not detected in any of the cell lines. RA treatment increased the levels of the three RAR mRNAs in most of the cell lines but had no effect on the RXR mRNAs. The CRABP-II mRNA level in SqCC/Y1 cells was lowest in cells grown in serum-free medium and increased when the cells were grown in medium with 5 or 10% serum. In contrast, the RXR-alpha mRNA level was inversely related to serum concentration. The results show that, in head and neck squamous cell carcinoma cells, there are no simple relationships among the expression of CRABPs, RARs, and RXRs and either squamous differentiation or response to RA-induced growth inhibition or suppression of squamous differentiation.

Isolation and characterization of putative intrinsic multidrug resistant Chinese hamster ovary cells by fluorescence activated cell sorting.

Most multidrug resistant cell lines reported in the literature were established by long-term continuous exposure of cells to stepwise increasing concentrations of antitumor drugs. However, these resistant cell lines may not be relevant to the majority of clinically resistant cells. In this study, we described the establishment of doxorubicin (Dox)-resistant Chinese hamster ovary cells by repeated flow cytometric cell sorting using the intrinsic fluorescence of Dox. In each sorting, the 15% least fluorescent cells were fractionated, grown to mass culture and sorted again. Results from a total of nine sorting cycles showed that the intracellular levels of Dox in the sorted cells were inversely proportional to the number of sorting cycles. The levels of P-glycoprotein mRNA in the sorted cells were increased, but reached a plateau of 2-3 fold after the fifth sorting cycle. The sorted cells exhibited a moderate but stable multidrug-resistant phenotype. Because the procedure involved minimal exposure of cells to the drug, the isolated cells are most likely related to naturally occurring (intrinsic) MDR cells.