Yong-Yu Liu | University of Louisiana at Monroe (original) (raw)

Papers by Yong-Yu Liu

Chemical Biology & Drug Design, 2014

The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibo... more The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibodies or a combination of antibodies with traditional chemotherapeutic agents or kinase inhibitors. Our approach is to target clinically validated HER2 domain IV with peptidomimetics and inhibit the protein-protein interactions (PPI) of HERs. Unlike antibodies, peptidomimetics have advantages in terms of stability, modification, and molecular size. We have designed peptidomimetics (compounds 5 and 9) that bind to HER2 domain IV, inhibit protein-protein interactions, and decrease cell viability in breast cancer cells with HER2 overexpression. We have shown, using enzyme fragment complementation and proximity ligation assays, that peptidomimetics inhibit the PPI of HER2:HER3. Compounds 5 and 9 suppressed the tumor growth in a xenograft mouse model. Furthermore, we have shown that these compounds inhibit PPI of HER2:HER3 and phosphorylation of HER2 as compared to control in tissue samples derived from in vivo studies. The stability of the compounds was also investigated in mouse serum, and the compounds exhibited stability with a half-life of up to 3 h. These results suggest that the novel peptidomimetics we have developed target the extracellular domain of HER2 protein and inhibit HER2: HER3 interaction, providing a novel method to treat HER2-positive cancer.

Cancer Research, 2011

Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by... more Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by inducing cell cycle arrest and apoptosis. Mutant p53 that is detected in over 50% cases of cancers not only loses its role in suppressing of tumor but also gains oncogenic function. Strategies to convert mutant p53 into wild-type of p53 have been suggested for cancer prevention and treatment, but they face a variety of challenges. Here we report an alternate approach that involves suppression of glucosylceramide synthase (GCS), an enzyme that glycosylates ceramide and blunts its pro-apoptotic activity in cancer cells. Human ovarian cancer cells expressing mutant p53 displayed resistance to apoptosis induced by DNA damage. We found that GCS silencing sensitized these mutant p53 cells to doxorubicin, but did not affect the sensitivity of cells with wild-type p53. GCS silencing increased the levels of phosphorylated p53 and p53-responsive genes including p21 Waf1/Cip1 , Bax and Puma, consistent with a redirection of the mutant p53 cells to apoptosis. Reactivated p53-dependent apoptosis was similarly verified in p53-mutant tumors where GCS was silenced. Inhibition of ceramide synthase with fumonisin B1 prevented p53 reactivation induced by GCS silencing, whereas addition of exogenous C6-ceramide reactivated p53 function in p53-mutant cells. Our findings indicate that restoring active ceramide to cells can resuscitate wild-type p53 function in p53 mutant cells, offering preclinical support for a novel type of mechanism-based therapy in the many human cancers harboring p53 mutations.

Molecular Cancer Therapeutics, 2008

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studi... more Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [ 3 H]palmitic acid showed that 4-HPR elicited a doseresponsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/ electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor D-erythro-N,N -dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR -induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther

Apoptosis, 2011

Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor ... more Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor p53, can translocate to mitochondria due to genotoxic stress. The mitochondrial targets of wt p53 have also been studied. However, whether mutant p53, which exists in 50% of human cancers, translocates to mitochondria and affects mitochondrial functions is unclear. In this study, we used doxorubicin, a chemotherapeutic drug, to treat five human lymphoma cell lines with wt, mutant or deficient in p53, to induce p53 activation and mitochondrial translocation. Our results demonstrated that mutant p53, like wt p53, was induced upon doxorubicin treatment. Similarly, a fraction of mutant p53 also translocated to mitochondria. However, Complex I and II activities in the mitochondria were compromised only in wt p53-bearing cells after doxorubicin treatment, but not in mutant p53-bearing cells. Similarly, doxorubicin treatment caused greater cell death only in wt p53-bearing cells, but not in mutant p53-bearing cells. When p53 deficient Ramos cells were transfected with mutant p53 (249S), the cells showed resistance to doxorubicin-induced cell death and decreases in complex activities. To reactivate mutant p53 and reverse chemoresistance, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) was used to treat mutant p53 cells.

... Yong-Yu Liu, Bret Taback, Jin Yuan Yu, Ping Xie, Valerie Gouaze, Armando E. Giuliano and Myle... more ... Yong-Yu Liu, Bret Taback, Jin Yuan Yu, Ping Xie, Valerie Gouaze, Armando E. Giuliano and Myles C. Cabot University of Louisiana at Monroe, Monroe, LA, Columbia University Medical Ctr., New York, NY, John Wayne Cancer Institute, Santa Monica, CA, Wuxi City No.1 ...

Partial hepatectomy results in activation of genes in the residual liver tissue which serve to re... more Partial hepatectomy results in activation of genes in the residual liver tissue which serve to restore glucose homeostasis and regenerate liver mass. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) is up-regulated following partial hepatectomy and IGFBP-1 can modulate both the metabolic and mitogenic effects of insulin-like growth factor-1 (IGF-I). The aim of the study was to compare the effects of partial hepatectomy on blood glucose levels and hepatic regeneration in wild-type and transgenic mice which constitutively overexpress IGFBP-1. Hepatic IGFBP-1 mRNA, blood glucose concentrations, liver mass and hepatic DNA synthesis were compared in sham-operated and partially hepatectomized transgenic and wild-type mice. Hepatic IGFBP-1 mRNA was higher in sham-operated transgenic than wild-type mice, but in both groups of mice, partial hepatectomy was associated with a significant rise in IGFBP-1 mRNA. The absolute decline in blood glucose levels following partial hepatectomy was greater in transgenic mice. Basal DNA synthesis and the response to IGF-I in isolated hepatocytes from both groups of mice were similar, and DNA synthesis in the regenerating liver in vivo was not significantly different in transgenic as compared to wild-type mice: 449.3 +/- 63.9 vs. 321.6 +/- 52.3 cpm/microgram DNA. Hepatic regeneration as measured by liver weight after hepatectomy was not different between transgenic and wild-type mice. Constitutive overexpression of IGFBP-1 does not enhance hepatic regeneration and does not prevent the decline in blood glucose following partial hepatectomy.

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzy... more Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.

Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduce... more Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduced P-glycoprotein that extrudes anticancer drugs from cells, is the most common mechanism detected in multidrug-resistant cancers. Direct measurement of cellular efflux of tumors in vivo, rather than estimation of MDR1 mRNA and P-glycoprotein levels in samples stored or embedded, can functionally characterize the mechanism of drug resistance and determine the choice of anticancer drugs for cancer patients. Herewith, we introduce a new approach to directly determine P-glycoprotein efflux of tumors. Employing Flutax-2 (Oregon green-488 paclitaxel) and fluorescence spectrophotometry, this method has successfully measured cellular transportability including efflux and accumulation in diverse cancer cell lines, tumors and other tissues with high reproducibility. With this method, we have quantitatively determined cellular efflux that is correlated with P-glycoprotein levels and the reversal effects of agents in cell lines of breast, ovarian, cervical and colon cancers, and in tumor-bearing mice. It has sensitively detected these alterations of P-glycoprotein efflux in approximately 5 mg tumor or other tissues with high confidence. This direct and quick functional assessment has a potential to determine drug resistance in different types of cancers after surgical resection. Further validation of this method in clinic settings for the diagnosis of drug resistance purpose is needed.

Journal of Lipid Research, 2004

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance ... more High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resis- tance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of an- tisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression

Biochemical Pharmacology, 2010

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide m... more Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer),

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mecha... more Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effec- tive treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and (3H)palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833)

Bioorganic & Medicinal Chemistry, 2014

Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evide... more Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with different backbones, in order to test the hypothesis that extending the conjugated system in ceramide analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy. The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylceramide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain intrinsic properties, ceramide analogs' pro-apoptotic activity is not disrupted by the normal drug-resistance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.

The international journal of biochemistry & cell biology, 2012

Myelosuppression and drug resistance are common adverse effects in cancer patients with chemother... more Myelosuppression and drug resistance are common adverse effects in cancer patients with chemotherapy, and those severely limit the therapeutic efficacy and lead treatment failure. It is unclear by which cellular mechanism anticancer drugs suppress bone marrow, while drug-resistant tumors survive. We report that due to the difference of glucosylceramide synthase (GCS), catalyzing ceramide glycosylation, doxorubicin (Dox) eliminates bone marrow stem cells (BMSCs) and expands breast cancer stem cells (BCSCs). It was found that Dox decreased the numbers of BMSCs (ABCG2(+)) and the sphere formation in a dose-dependent fashion in isolated bone marrow cells. In tumor-bearing mice, Dox treatments (5mg/kg, 6 days) decreased the numbers of BMSCs and white blood cells; conversely, those treatments increased the numbers of BCSCs (CD24(-)/CD44(+)/ESA(+)) more than threefold in the same mice. Furthermore, therapeutic-dose of Dox (1mg/kg/week, 42 days) decreased the numbers of BMSCs while it incre...

Methods in molecular biology (Clifton, N.J.), 2011

Flow cytometry can sensitively detect and efficiently sort cells based on fluorescent signals int... more Flow cytometry can sensitively detect and efficiently sort cells based on fluorescent signals integrated into cellular markers of proteins or DNA. It has been broadly applied to assess cell division, apoptosis and to isolate cells including stem cells. As the seeds for tumorigenesis and metastasis, cancer stem cells (CSCs) are often more resistant to cytotoxins and anticancer agents than other heterogeneous cells in tumors. Analyzing CSCs under treatments is an effective way to evaluate new therapeutic agents for cancers. We introduce a method using flow cytometry to assess breast CSCs (CD44(+)/CD24(-)/(low)) in human MCF-7/Dox breast cancer cells, after the treatment of mixed-backbone oligonucleotide against glucosylceramide synthase. Flow cytometry analysis of CSCs is a reliable, effective, and easy-handling approach to screen agents targeting CSCs.

International journal of pharmaceutics, Jan 15, 2012

The objective of this study was to increase the potency of doxorubicin against adriamycin-resista... more The objective of this study was to increase the potency of doxorubicin against adriamycin-resistant NCI/ADR-RES cells by concurrent treatment with doxorubicin and MBO-asGCS loaded solid-lipid nanoparticles (SLN). Loading doxorubicin as ion-pair complex with deoxytaurocholate into cationic and neutral SLN was investigated. Fast release and poor entrapment were observed in cationic nanoparticles, which were corrected by entrapping the complex in neutral polyoxyethylene (20) stearyl ether (Brij(®) 78)/VitE-TPGS nanoparticles. Slow doxorubicin release confirmed the influence of charge and electrolytes on the dissociation of ion-pair complexes. To evaluate antitumor activity, NCI/ADR-RES cells were treated with separate SLN: one loaded with doxorubicin and another carrying MBO-asGCS oligonucleotide. The viability of cells treated with 5 μM doxorubicin was reduced to 17.2% whereas viability was reduced to 2.5% for cells treated with both 5 μM doxorubicin SLN and 100 nM MBO-asGCS SLN. This...

Cancer Research, 2011

Mutant p53 is frequently detected in cancers in which p53 has lost its ability in tumor suppressi... more Mutant p53 is frequently detected in cancers in which p53 has lost its ability in tumor suppression and gained function in promoting tumor progression. Restoration of p53 functions by replacement of wild-type p53 and inhibition of its degradation or increment of its transcriptional activity has been applied to the prevention and treatment of cancers. Recent evidence indicates that disrupting ceramide glycosylation can resuscitate wild-type p53 expression and p53-dependent apoptosis in mutant p53 tumors. A posttranscriptional process that can turn on wild-type p53 expression and abrogate mutant p53 may provide a new strategy to eradicate mutant p53 cancers. Cancer Res; 71(20); 6295-9. Ó2011 AACR.

Chemical Biology & Drug Design, 2014

The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibo... more The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibodies or a combination of antibodies with traditional chemotherapeutic agents or kinase inhibitors. Our approach is to target clinically validated HER2 domain IV with peptidomimetics and inhibit the protein-protein interactions (PPI) of HERs. Unlike antibodies, peptidomimetics have advantages in terms of stability, modification, and molecular size. We have designed peptidomimetics (compounds 5 and 9) that bind to HER2 domain IV, inhibit protein-protein interactions, and decrease cell viability in breast cancer cells with HER2 overexpression. We have shown, using enzyme fragment complementation and proximity ligation assays, that peptidomimetics inhibit the PPI of HER2:HER3. Compounds 5 and 9 suppressed the tumor growth in a xenograft mouse model. Furthermore, we have shown that these compounds inhibit PPI of HER2:HER3 and phosphorylation of HER2 as compared to control in tissue samples derived from in vivo studies. The stability of the compounds was also investigated in mouse serum, and the compounds exhibited stability with a half-life of up to 3 h. These results suggest that the novel peptidomimetics we have developed target the extracellular domain of HER2 protein and inhibit HER2: HER3 interaction, providing a novel method to treat HER2-positive cancer.

Cancer Research, 2011

Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by... more Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by inducing cell cycle arrest and apoptosis. Mutant p53 that is detected in over 50% cases of cancers not only loses its role in suppressing of tumor but also gains oncogenic function. Strategies to convert mutant p53 into wild-type of p53 have been suggested for cancer prevention and treatment, but they face a variety of challenges. Here we report an alternate approach that involves suppression of glucosylceramide synthase (GCS), an enzyme that glycosylates ceramide and blunts its pro-apoptotic activity in cancer cells. Human ovarian cancer cells expressing mutant p53 displayed resistance to apoptosis induced by DNA damage. We found that GCS silencing sensitized these mutant p53 cells to doxorubicin, but did not affect the sensitivity of cells with wild-type p53. GCS silencing increased the levels of phosphorylated p53 and p53-responsive genes including p21 Waf1/Cip1 , Bax and Puma, consistent with a redirection of the mutant p53 cells to apoptosis. Reactivated p53-dependent apoptosis was similarly verified in p53-mutant tumors where GCS was silenced. Inhibition of ceramide synthase with fumonisin B1 prevented p53 reactivation induced by GCS silencing, whereas addition of exogenous C6-ceramide reactivated p53 function in p53-mutant cells. Our findings indicate that restoring active ceramide to cells can resuscitate wild-type p53 function in p53 mutant cells, offering preclinical support for a novel type of mechanism-based therapy in the many human cancers harboring p53 mutations.

Molecular Cancer Therapeutics, 2008

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studi... more Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [ 3 H]palmitic acid showed that 4-HPR elicited a doseresponsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/ electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor D-erythro-N,N -dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR -induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther

Apoptosis, 2011

Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor ... more Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor p53, can translocate to mitochondria due to genotoxic stress. The mitochondrial targets of wt p53 have also been studied. However, whether mutant p53, which exists in 50% of human cancers, translocates to mitochondria and affects mitochondrial functions is unclear. In this study, we used doxorubicin, a chemotherapeutic drug, to treat five human lymphoma cell lines with wt, mutant or deficient in p53, to induce p53 activation and mitochondrial translocation. Our results demonstrated that mutant p53, like wt p53, was induced upon doxorubicin treatment. Similarly, a fraction of mutant p53 also translocated to mitochondria. However, Complex I and II activities in the mitochondria were compromised only in wt p53-bearing cells after doxorubicin treatment, but not in mutant p53-bearing cells. Similarly, doxorubicin treatment caused greater cell death only in wt p53-bearing cells, but not in mutant p53-bearing cells. When p53 deficient Ramos cells were transfected with mutant p53 (249S), the cells showed resistance to doxorubicin-induced cell death and decreases in complex activities. To reactivate mutant p53 and reverse chemoresistance, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) was used to treat mutant p53 cells.

... Yong-Yu Liu, Bret Taback, Jin Yuan Yu, Ping Xie, Valerie Gouaze, Armando E. Giuliano and Myle... more ... Yong-Yu Liu, Bret Taback, Jin Yuan Yu, Ping Xie, Valerie Gouaze, Armando E. Giuliano and Myles C. Cabot University of Louisiana at Monroe, Monroe, LA, Columbia University Medical Ctr., New York, NY, John Wayne Cancer Institute, Santa Monica, CA, Wuxi City No.1 ...

Partial hepatectomy results in activation of genes in the residual liver tissue which serve to re... more Partial hepatectomy results in activation of genes in the residual liver tissue which serve to restore glucose homeostasis and regenerate liver mass. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) is up-regulated following partial hepatectomy and IGFBP-1 can modulate both the metabolic and mitogenic effects of insulin-like growth factor-1 (IGF-I). The aim of the study was to compare the effects of partial hepatectomy on blood glucose levels and hepatic regeneration in wild-type and transgenic mice which constitutively overexpress IGFBP-1. Hepatic IGFBP-1 mRNA, blood glucose concentrations, liver mass and hepatic DNA synthesis were compared in sham-operated and partially hepatectomized transgenic and wild-type mice. Hepatic IGFBP-1 mRNA was higher in sham-operated transgenic than wild-type mice, but in both groups of mice, partial hepatectomy was associated with a significant rise in IGFBP-1 mRNA. The absolute decline in blood glucose levels following partial hepatectomy was greater in transgenic mice. Basal DNA synthesis and the response to IGF-I in isolated hepatocytes from both groups of mice were similar, and DNA synthesis in the regenerating liver in vivo was not significantly different in transgenic as compared to wild-type mice: 449.3 +/- 63.9 vs. 321.6 +/- 52.3 cpm/microgram DNA. Hepatic regeneration as measured by liver weight after hepatectomy was not different between transgenic and wild-type mice. Constitutive overexpression of IGFBP-1 does not enhance hepatic regeneration and does not prevent the decline in blood glucose following partial hepatectomy.

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzy... more Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.

Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduce... more Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduced P-glycoprotein that extrudes anticancer drugs from cells, is the most common mechanism detected in multidrug-resistant cancers. Direct measurement of cellular efflux of tumors in vivo, rather than estimation of MDR1 mRNA and P-glycoprotein levels in samples stored or embedded, can functionally characterize the mechanism of drug resistance and determine the choice of anticancer drugs for cancer patients. Herewith, we introduce a new approach to directly determine P-glycoprotein efflux of tumors. Employing Flutax-2 (Oregon green-488 paclitaxel) and fluorescence spectrophotometry, this method has successfully measured cellular transportability including efflux and accumulation in diverse cancer cell lines, tumors and other tissues with high reproducibility. With this method, we have quantitatively determined cellular efflux that is correlated with P-glycoprotein levels and the reversal effects of agents in cell lines of breast, ovarian, cervical and colon cancers, and in tumor-bearing mice. It has sensitively detected these alterations of P-glycoprotein efflux in approximately 5 mg tumor or other tissues with high confidence. This direct and quick functional assessment has a potential to determine drug resistance in different types of cancers after surgical resection. Further validation of this method in clinic settings for the diagnosis of drug resistance purpose is needed.

Journal of Lipid Research, 2004

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance ... more High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resis- tance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of an- tisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression

Biochemical Pharmacology, 2010

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide m... more Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer),

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mecha... more Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effec- tive treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and (3H)palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833)

Bioorganic & Medicinal Chemistry, 2014

Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evide... more Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with different backbones, in order to test the hypothesis that extending the conjugated system in ceramide analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy. The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylceramide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain intrinsic properties, ceramide analogs' pro-apoptotic activity is not disrupted by the normal drug-resistance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.

The international journal of biochemistry & cell biology, 2012

Myelosuppression and drug resistance are common adverse effects in cancer patients with chemother... more Myelosuppression and drug resistance are common adverse effects in cancer patients with chemotherapy, and those severely limit the therapeutic efficacy and lead treatment failure. It is unclear by which cellular mechanism anticancer drugs suppress bone marrow, while drug-resistant tumors survive. We report that due to the difference of glucosylceramide synthase (GCS), catalyzing ceramide glycosylation, doxorubicin (Dox) eliminates bone marrow stem cells (BMSCs) and expands breast cancer stem cells (BCSCs). It was found that Dox decreased the numbers of BMSCs (ABCG2(+)) and the sphere formation in a dose-dependent fashion in isolated bone marrow cells. In tumor-bearing mice, Dox treatments (5mg/kg, 6 days) decreased the numbers of BMSCs and white blood cells; conversely, those treatments increased the numbers of BCSCs (CD24(-)/CD44(+)/ESA(+)) more than threefold in the same mice. Furthermore, therapeutic-dose of Dox (1mg/kg/week, 42 days) decreased the numbers of BMSCs while it incre...

Methods in molecular biology (Clifton, N.J.), 2011

Flow cytometry can sensitively detect and efficiently sort cells based on fluorescent signals int... more Flow cytometry can sensitively detect and efficiently sort cells based on fluorescent signals integrated into cellular markers of proteins or DNA. It has been broadly applied to assess cell division, apoptosis and to isolate cells including stem cells. As the seeds for tumorigenesis and metastasis, cancer stem cells (CSCs) are often more resistant to cytotoxins and anticancer agents than other heterogeneous cells in tumors. Analyzing CSCs under treatments is an effective way to evaluate new therapeutic agents for cancers. We introduce a method using flow cytometry to assess breast CSCs (CD44(+)/CD24(-)/(low)) in human MCF-7/Dox breast cancer cells, after the treatment of mixed-backbone oligonucleotide against glucosylceramide synthase. Flow cytometry analysis of CSCs is a reliable, effective, and easy-handling approach to screen agents targeting CSCs.

International journal of pharmaceutics, Jan 15, 2012

The objective of this study was to increase the potency of doxorubicin against adriamycin-resista... more The objective of this study was to increase the potency of doxorubicin against adriamycin-resistant NCI/ADR-RES cells by concurrent treatment with doxorubicin and MBO-asGCS loaded solid-lipid nanoparticles (SLN). Loading doxorubicin as ion-pair complex with deoxytaurocholate into cationic and neutral SLN was investigated. Fast release and poor entrapment were observed in cationic nanoparticles, which were corrected by entrapping the complex in neutral polyoxyethylene (20) stearyl ether (Brij(®) 78)/VitE-TPGS nanoparticles. Slow doxorubicin release confirmed the influence of charge and electrolytes on the dissociation of ion-pair complexes. To evaluate antitumor activity, NCI/ADR-RES cells were treated with separate SLN: one loaded with doxorubicin and another carrying MBO-asGCS oligonucleotide. The viability of cells treated with 5 μM doxorubicin was reduced to 17.2% whereas viability was reduced to 2.5% for cells treated with both 5 μM doxorubicin SLN and 100 nM MBO-asGCS SLN. This...

Cancer Research, 2011

Mutant p53 is frequently detected in cancers in which p53 has lost its ability in tumor suppressi... more Mutant p53 is frequently detected in cancers in which p53 has lost its ability in tumor suppression and gained function in promoting tumor progression. Restoration of p53 functions by replacement of wild-type p53 and inhibition of its degradation or increment of its transcriptional activity has been applied to the prevention and treatment of cancers. Recent evidence indicates that disrupting ceramide glycosylation can resuscitate wild-type p53 expression and p53-dependent apoptosis in mutant p53 tumors. A posttranscriptional process that can turn on wild-type p53 expression and abrogate mutant p53 may provide a new strategy to eradicate mutant p53 cancers. Cancer Res; 71(20); 6295-9. Ó2011 AACR.