Browsing by Author "Ahmad Ali S Alghamdi"

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A Role for GRP78 in the Actions of IGFBP-2 and -3 on Breast Cancer Cells
Ahmad Ali S Alghamdi; Jeffery Holly
Glucose-regulated protein 78 (GRP78), a member of the heat shock protein family that functions as a master regulator of the endoplasmic reticulum (ER) stress pathway (the unfolded protein response (UPR)), has been heavily implicated in many cellular processes, including cancer cell proliferation, survival and metastasis. GRP78 have been identified as a binding partner for IGFBP-3, this association of IGFBP-3 with GRP78 culminated in opposite effects on cell function, survival, and apoptosis. We have previously shown that both IGFs binding proteins; IGFBP-2 and-3, which act mainly as carriers for IGFs in the circulation, are implicated in affecting cancer cell survival and growth in a context-dependent manner. I aimed to assess if GRP78 has a role on the physiological functions of IGFBP-2 and IGFBP-3 and and whether an interaction with GRP78 was required for their actions. I found that adding exogenous IGFBP-3 to ERα -ve cells caused an increase in the levels of GRP78, that was associated with a decrease in cell growth and an accentuation of chemotherapy-induced apoptosis. Silencing GRP78 in ERα-negative cells blocked the anti-proliferative effect of IGFBP-3 and switched the actions of IGFBP-3 from being pro-apoptotic to acting as a survival factor. Also, I found that GRP78 silencing switched the effect of IGFBP-3 on cellular invasion from inhibiting to promoting. Silencing IGFBP-2 in ERα +ve cells using siRNA led to a reduction in GRP78 that was associated with a decrease in cell invasion and conversely silencing GRP78 led to a reduction in IGFBP-2. Adding exogenous IGFBP-2 to ERα +ve cells caused a GRP78-independent decrease in cell growth, an increase in the levels of GRP78 that was associated with an increase in the RGD motif-mediated cellular migration. Silencing GRP78 in ERα +ve cells blocked the migratory effect of IGFBP-2. Using immunoprecipitation, we confirmed that IGFBP-2 and IGFBP-3 each bound to GRP78 in whole-cell lysates. Localisation studies showed that both IGFBP-2 and -3 localise with GRP78 on the cytoplasm and the nucleus. Moreover, biotinylation and avidin pull-down studies showed that exogenous IGFBP-2 and -3 alter the abundance of GRP78 on the cell surface. The immunohistochemistry data showed a direct correlation between the expression of GRP78 and IGFBPs -2 and -3 in breast cancer tumour sections. Also showed a negative correlation between PTEN and IGFBP-2 with no correlation between PTEN and GRP78. Kaplan-Meier survival plots revealed that patients with low GRP78 expression that are positive for IGFBP-3 had poorer survival rates than those with low IGFBP-3 levels. It also showed that PTEN negativity and PTEN loss correlate negatively with patients’ survival. These data suggest that both IGFBP-2 and IGFBP-3 interact with GRP78 and the effects of both binding proteins on breast cancer cells are influenced by the presence of GRP78.
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HPLC with electrochemical detection for the determination of some Benzodiazepine drugs in Biological fluids
(Saudi Digital Library) Ahmad Ali S Alghamdi; Professor John Hart
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