Xin Gu

Protandim, a Fundamentally New Antioxidant Approach in Chemoprevention Using Mouse Two-Stage Skin Carcinogenesis as a Model

Oxidative stress is an important contributor to cancer development. Consistent with that, antioxidant enzymes have been demonstrated to suppress tumorigenesis when being elevated both in vitro and in vivo, making induction of these enzymes a more potent approach for cancer prevention. Protandim, a well-defined combination of widely studied medicinal plants, has been shown to induce superoxide dismutase (SOD) and catalase activities and reduce superoxide generation and lipid peroxidation in healthy human subjects. To investigate whether Protandim can suppress tumor formation by a dietary approach, a two-stage mouse skin carcinogenesis study was performed. At the end of the study, the mice on a Protandim-containing basal diet had similar body weight compared with those on the basal diet, which indicated no overt toxicity by Protandim. After three weeks on the diets, there was a significant increase in the expression levels of SOD and catalase, in addition to the increases in SOD activities. Importantly, at the end of the carcinogenesis study, both skin tumor incidence and multiplicity were reduced in the mice on the Protandim diet by 33% and 57% respectively, compared with those on basal diet. Biochemical and histological studies revealed that the Protandim diet suppressed tumor promoter-induced oxidative stress (evidenced by reduction of protein carbonyl levels), cell proliferation (evidenced by reduction of skin hyperplasia and suppression of PKC/JNK/Jun pathway), and inflammation (evidenced by reduction of ICAM-1/VCAM-1 expression, NF-κB binding activity, and nuclear p65/p50 levels). Overall, induction of antioxidant enzymes by Protandim may serve as a practical and potent approach for cancer prevention.

Suppression of Glucosylceramide Synthase Restores p53-Dependent Apoptosis in Mutant p53 Cancer Cells

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 more than 50% of cases of cancers loses its role in suppression of tumors but gains in oncogenic function. Strategies to convert mutant p53 into wild-type p53 have been suggested for cancer prevention and treatment, but they face a variety of challenges. Here, we report an alternative approach that involves suppression of glucosylceramide synthase (GCS), an enzyme that glycosylates ceramide and blunts its proapoptotic 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.

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Background: Glucosylceramide synthase catalyzes ceramide glycosylation that regulates the synthesis of glycosphingolipids. Results: Increased globo-series glycosphingolipids in breast cancer stem cells activate c-Src signaling and β-catenin-mediated transcription up-regulating stem cell factors. Conclusion: Ceramide glycosylation maintains the stemness of cancer stem cells. Significance: Glycosphingolipids in cell membrane actively participate in maintaining cancer stem cells. Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44+/ESA+/CD24− markers, Gb3 activates c-Src/β-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/β-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.

Glucosylceramide synthase, a factor in modulating drug resistance, is overexpressed in metastatic breast carcinoma

Drug resistance causes treatment failure in approximately 50% of breast cancer patients with chemotherapy. Overexpression of glucosylceramide synthase (GCS) confers drug resistance in cancer cells, and suppression of GCS sensitizes cancers to chemotherapy in preclinical studies. Thus, GCS becomes a potential target to reverse drug resistance; however, little is known about GCS expression levels in normal tissues and whether GCS overexpression is associated with metastatic cancers. Herewith, we report our studies in GCS expression levels and breast cancer from patients. GCS levels were analyzed using cancer profiling arrays, breast cancer histo-arrays and quantitative RT-PCR in tumor tissues. We found that breast (18 exp. index) and other hormone-dependent organs (testis, cervix, ovary, prostate) displayed the lowest levels of GCS mRNA, whereas liver (52 exp. index) and other organs (kidney, bladder, stomach) displayed the highest levels of GCS. GCS mRNA levels were significantly elevated in tumors of breast, cervix, rectum and small intestine, as compared to each paired normal tissue. In mammary tissue, GCS overexpression was detected in breast cancers with metastasis, but not in benign fibroadenoma or primary tumors. GCS overexpression was coincident with HER2 expression (γ2=0.84) in ER-negative breast adenocarcinoma. In tumor specimens, GCS mRNA was elevated by 4-fold and significantly associated with stage III (5/7), lymph node-positive (7/8) and estrogen receptor-positive breast cancers (7/9). GCS expression was significantly and selectively elevated in breast cancer, in particular in metastatic disease. GCS overexpression was highly associated with ER-positive and HER2-positive breast cancer with metastasis. Although a small study, these data suggest that GCS may be a prognostic indicator and potential target for the treatment of chemotherapy-refractory breast cancer.

Blocking mitochondrial permeability transition prevents p53 mitochondrial translocation during skin tumor promotion

The tumor suppressor p53 can translocate into mitochondria and activate apoptosis. Here we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by blocking mitochondrial permeability transition pore using cyclosporine A (CsA) and bongkrekic acid (BA) in skin epidermal JB6 cells and skin tissues. Our results demonstrated that CsA and BA blocked TPA‐induced p53 translocation, leading to protection against the loss of mitochondrial membrane potential and Complex I activity, and eventually suppression of apoptosis. Thus, our results suggest that mitochondrial permeability transition is required for p53 mitochondrial translocation.

Immunoglobulin Heavy Chain Can Be Amyloidogenic: Morphologic Characterization Including Immunoelectron Microscopy

It is not well recognized that monoclonal immunoglobulin heavy chains or their fragments can be amyloidogenic. Amyloidosis due to heavy chains, referred to as AH amyloidosis, is rare with only three cases previously reported. An additional case of AH amyloidosis is reported. To the best of our knowledge, this is the first case of IgM heavy chain amyloidosis. A 59-year-old man presented with nephrotic syndrome. Immunoelectrophoresis detected a monoclonal IgM lambda in his serum and free lambda light chains in his urine. A renal biopsy showed amyloidosis, in which the amyloid deposits stained only for mu heavy chain by immunofluorescent and immunoelectron microscopic studies. This case suggests that monoclonal immunoglobulin heavy chains can be amyloidogenic. Furthermore, this type of amyloidosis shares the same morphologic features with other types of amyloidosis and can involve the kidney to produce nephrotic syndrome.

Laser‐capture microdissection of oropharyngeal epithelium indicates restriction of Epstein–Barr virus receptor/CD21 mRNA to tonsil epithelial cells

BACKGROUNDnEpstein-Barr virus colonizes the oropharynx of a majority of individuals. It infects B lymphocytes and epithelial cells and can contribute to the development of both lymphoid and epithelial tumors. The virus uses CD21 for attachment to B cells which constitutively express the protein. Infection of epithelial cells in vitro is also more efficient if CD21 is available. However, its potential contribution to infection in vivo has been difficult to evaluate as discrepant results with antibodies have made it difficult to determine which, if any, epithelial cells in the oropharynx express CD21.nnnMETHODSnTo reevaluate CD21 expression by an alternative method, epithelial cells were isolated by laser-capture microdissection from formalin-fixed sections of tissues from various parts of the oropharynx and mRNA was amplified with primers specific for the exons of CD21 which code for the Epstein-Barr virus binding site.nnnRESULTSnCD21 mRNA was expressed in tonsil epithelium, but not in epithelium from buccal mucosa, uvula, soft palate or tongue.nnnCONCLUSIONSnCD21 does not contribute to infection of most normal epithelial tissues in the oropharynx, but may contribute to infection of epithelial cells in the tonsil, where virus has been demonstrated in healthy carriers.

The Chemopreventive Effects of Protandim: Modulation of p53 Mitochondrial Translocation and Apoptosis during Skin Carcinogenesis

Protandim, a well defined dietary combination of 5 well-established medicinal plants, is known to induce endogenous antioxidant enzymes, such as manganese superoxide dismutase (MnSOD). Our previous studies have shown through the induction of various antioxidant enzymes, products of oxidative damage can be decreased. In addition, we have shown that tumor multiplicity and incidence can be decreased through the dietary administration of Protandim in the two-stage skin carcinogenesis mouse model. It has been demonstrated that cell proliferation is accommodated by cell death during DMBA/TPA treatment in the two-stage skin carcinogenesis model. Therefore, we investigated the effects of the Protandim diet on apoptosis; and proposed a novel mechanism of chemoprevention utilized by the Protandim dietary combination. Interestingly, Protandim suppressed DMBA/TPA induced cutaneous apoptosis. Recently, more attention has been focused on transcription-independent mechanisms of the tumor suppressor, p53, that mediate apoptosis. It is known that cytoplasmic p53 rapidly translocates to the mitochondria in response to pro-apoptotic stress. Our results showed that Protandim suppressed the mitochondrial translocation of p53 and mitochondrial outer membrane proteins such as Bax. We examined the levels of p53 and MnSOD expression/activity in murine skin JB6 promotion sensitive (P+) and promotion-resistant (P-) epidermal cells. Interestingly, p53 was induced only in P+ cells, not P- cells; whereas MnSOD is highly expressed in P- cells when compared to P+ cells. In addition, wild-type p53 was transfected into JB6 P- cells. We found that the introduction of wild-type p53 promoted transformation in JB6 P- cells. Our results suggest that suppression of p53 and induction of MnSOD may play an important role in the tumor suppressive activity of Protandim.

Ultrastructural Changes in Cerebral Capillary Pericytes in Aged Notch3 Mutant Transgenic Mice

Pericytes, the specialized vascular smooth muscle cells (VSMCs), play an important role in supporting and maintaining the structure of capillaries. Pericytes show biochemical and physiologic features similar to VSMC, usually containing smooth muscle actin fibers and rich endoplasm reticulum. Studies have indicated that degeneration of VSMCs due to Notch3 mutations is the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, it remains unclear whether the Notch3 mutation also affects cerebral cortex capillary pericytes. In this ultrastructural morphologic study, the authors have observed pathological changes in the cerebral cortex capillary pericytes in aged mice that carry human mutant Notch3 genes. The number of abnormal pericytes in the cerebral cortex in Notch3 gene mutant mice was slightly increased when compared to an age-matched control group. Morphologically, the pericytes in the brains of Notch3 gene mutant mice showed more severe cellular injury, such as the presence of damaged mitochondria, secondary lysosomes, and large cytoplasmic vesicles. In addition, morphologic structures related to autophagy were also present in the pericytes of Notch3 gene mutant group. These ultrastructural morphologic alterations suggest that Notch3 mutation precipitates age-related pericytic degeneration that might result in cellular injury and trigger autophagic apoptosis. Microvascular dysfunction due to pericyte degeneration could initiate secondary neurodegenerative changes in brain parenchyma. These findings provide new insight into understanding the role of pericyte degeneration in the phathogenesis of CADASIL disease.

Direct assessment of P-glycoprotein efflux to determine tumor response to chemotherapy.

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 5mg 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.