Use of Remote Regulatory Assessment (RRA) for Site Evaluations during the COVID-19 Pandemic: The USFDA's Office of Study Integrity and Surveillance (OSIS) Perspective.

Travel restrictions during the novel coronavirus, SARS-CoV-2 (COVID-19) public health emergency affected the U.S. Food and Drug Administration's (FDA) ability to conduct on-site bioavailability/bioequivalence (BA/BE) and Good Laboratory Practice (GLP) nonclinical inspections.

Sildenafil (Viagra) sensitizes prostate cancer cells to doxorubicin-mediated apoptosis through CD95.

We previously reported that Sildenafil enhances apoptosis and antitumor efficacy of doxorubicin (DOX) while attenuating its cardiotoxic effect in prostate cancer. In the present study, we investigated the mechanism by which sildenafil sensitizes DOX in killing of prostate cancer (PCa) cells, DU145. The death receptor Fas (APO-1 or CD95) induces apoptosis in many carcinoma cells, which is negatively regulated by anti-apoptotic molecules such as FLIP (Fas-associated death domain (FADD) interleukin-1-converting enzyme (FLICE)-like inhibitory protein).

Simultaneous exposure of transformed cells to SRC family inhibitors and CHK1 inhibitors causes cell death.

The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01 (7-hydroxystaurosporine), AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor -induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC.

A serotype 5/3 adenovirus expressing MDA-7/IL-24 infects renal carcinoma cells and promotes toxicity of agents that increase ROS and ceramide levels.

Agents that generate reactive oxygen species (ROS) are recognized to enhance MDA-7/IL-24 lethality. The present studies focused on clarifying how such agents enhanced MDA-7/IL-24 toxicity in renal cell carcinoma cells (RCCs). Infection of RCCs with a tropism-modified serotype 5/3 adenovirus expressing MDA-7/IL-24 (Ad.

Sildenafil increases chemotherapeutic efficacy of doxorubicin in prostate cancer and ameliorates cardiac dysfunction.

We have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor sildenafil (Viagra) induces a powerful effect on reduction of infarct size following ischemia/reperfusion injury and improvement of left ventricular dysfunction in the failing heart after myocardial infarction or doxorubicin (DOX) treatment. In the present study, we further investigated the potential effects of sildenafil on improving antitumor efficacy of DOX in prostate cancer. Cotreatment with sildenafil enhanced DOX-induced apoptosis in PC-3 and DU145 prostate cancer cells, which was mediated by enhanced generation of reactive oxygen species, up-regulation of caspase-3 and caspase-9 activities, reduced expression of Bcl-xL, and phosphorylation of Bad.

Inhibition of MCL-1 in breast cancer cells promotes cell death in vitro and in vivo.

The present studies have examined approaches to suppress MCL-1 function in breast cancer cells, as a means to promote tumor cell death. Treatment of breast cancer cells with CDK inhibitors (flavopiridol; roscovitine) enhanced the lethality of the ERBB1 inhibitor lapatinib in a synergistic fashion. CDK inhibitors interacted with lapatinib to reduce MCL-1 expression and over-expression of MCL-1 or knock down of BAX and BAK suppressed drug combination lethality.

Poly(ADP-ribose) polymerase 1 modulates the lethality of CHK1 inhibitors in carcinoma cells.

Prior studies have demonstrated that inhibition of CHK1 can promote the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphorylation of histone H2AX and that inhibition of poly(ADP-ribose) polymerase 1 (PARP1) can affect growth factor-induced ERK1/2 activation. The present studies were initiated to determine whether CHK1 inhibitors interacted with PARP1 inhibition to facilitate apoptosis. Transient expression of dominant-negative CHK1 raised basal ERK1/2 activity and prevented CHK1 inhibitors from activating ERK1/2.

Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca(2+)-de novo ceramide-PP2A-reactive oxygen species-dependent signaling pathway.

The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca(2+), and ceramide levels in gastrointestinal tumor cells.

17-allylamino-17-demethoxygeldanamycin and MEK1/2 inhibitors kill GI tumor cells via Ca2+-dependent suppression of GRP78/BiP and induction of ceramide and reactive oxygen species.

The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca(2+) levels and reduced GRP78/BiP expression in a Ca(2+)-dependent manner.

Role of oxidative stress and MAPK signaling in reference moist smokeless tobacco-induced HOK-16B cell death.

The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3h of exposure.

PERK-dependent regulation of ceramide synthase 6 and thioredoxin play a key role in mda-7/IL-24-induced killing of primary human glioblastoma multiforme cells.

Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors.

Inhibition of MCL-1 enhances lapatinib toxicity and overcomes lapatinib resistance via BAK-dependent autophagy.

Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-X(L)/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells.

Targeting sphingosine kinase 1 inhibits Akt signaling, induces apoptosis, and suppresses growth of human glioblastoma cells and xenografts.

Sphingosine-1-phosphate is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce sphingosine-1-phosphate, is up-regulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and nonestablished human GBM6 cells.

BCL-2 family inhibitors enhance histone deacetylase inhibitor and sorafenib lethality via autophagy and overcome blockade of the extrinsic pathway to facilitate killing.

We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI).

Sorafenib and vorinostat kill colon cancer cells by CD95-dependent and -independent mechanisms.

We examined the interaction between the multikinase inhibitor sorafenib and histone deacetylase inhibitors. Sorafenib and vorinostat synergized (sorafenib + vorinostat) to kill HCT116 and SW480 cells. In SW480 cells, sorafenib + vorinostat increased CD95 plasma membrane levels and promoted death-inducing signal complex (DISC) formation, and drug toxicity was blocked by knockdown of CD95 or overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s).

Autophagy and the functional roles of Atg5 and beclin-1 in the anti-tumor effects of 3beta androstene 17alpha diol neuro-steroid on malignant glioma cells.

In this study, we demonstrate that the anti-tumor activity of the neuro-steroid, 3beta androstene 17alpha diol (17alpha-AED) on malignant glioma cells is mediated by the induction of autophagy. 17alpha-AED can inhibit the proliferation an induce cell death of multiple, unrelated gliomas with an IC(50) between 8 and 25muM. 17alpha-AED treatment induced the formation of autophagosomes and acidic vesicular organelles in human malignant gliomas which was blocked by bafilomycin A1 or 3-methyladenine.

Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic Ras is a dominant predictor for resistance.

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866.

Mitogen-activated protein kinase kinase 1/2 inhibitors and 17-allylamino-17-demethoxygeldanamycin synergize to kill human gastrointestinal tumor cells in vitro via suppression of c-FLIP-s levels and activation of CD95.

Prior studies have noted that inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors [PD184352; AZD6244 (ARRY-142886)] interacted in a synergistic manner with geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin] to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of extracellular signal-regulated kinase 1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was reactive oxygen species dependent.

Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation.

We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8.

Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation.

Purpose And Design: Mechanism(s) by which the multikinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal, and pancreatic adenocarcinoma cells has been defined.

Results: Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal, and pancreatic adenocarcinoma cells in multiple short-term viability (24-96 h) and in long-term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase-8 and, to a lesser extent, by inhibition of caspase-9.

Multiple cyclin kinase inhibitors promote bile acid-induced apoptosis and autophagy in primary hepatocytes via p53-CD95-dependent signaling.

Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95.

Lapatinib resistance in HCT116 cells is mediated by elevated MCL-1 expression and decreased BAK activation and not by ERBB receptor kinase mutation.

We have defined some of the mechanisms by which the kinase inhibitor lapatinib kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2, extracellular signal-regulated kinases 1/2, and AKT, and radiosensitized HCT116 cells. Prolonged incubation of HCT116 cells with lapatinib caused cell killing followed by outgrowth of lapatinib-adapted cells.

Human chorionic gonadotropin (hCG) interacts with lovastatin and ionizing radiation to modulate prostate cancer cell viability in vivo.

The ability of human chorionic gonadotropin (hCG) to modify prostate carcinoma viability in vitro and in vivo when combined with the HMG CoA reductase inhibitor lovastatin and ionizing radiation was investigated. Treatment of PC-3 cells in vitro with hCG caused a modest increase in numbers of non-viable cells within 96 h. Treatment of cells with hCG followed by exposure to the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG.

Transient exposure of carcinoma cells to RAS/MEK inhibitors and UCN-01 causes cell death in vitro and in vivo.

The present studies were initiated to determine in greater molecular detail how MEK1/2 inhibitors [PD184352 and AZD6244 (ARRY-142886)] interact with UCN-01 (7-hydroxystaurosporine) to kill mammary carcinoma cells in vitro and radiosensitize mammary tumors in vitro and in vivo and whether farnesyl transferase inhibitors interact with UCN-01 to kill mammary carcinoma cells in vitro and in vivo. Expression of constitutively activated MEK1 EE or molecular suppression of JNK and p38 pathway signaling blocked MEK1/2 inhibitor and UCN-01 lethality, effects dependent on the expression of BAX, BAK, and, to a lesser extent, BIM and BID. In vitro colony formation studies showed that UCN-01 interacted synergistically with the MEK1/2 inhibitors PD184352 or AZD6244 and the farnesyl transferase inhibitors FTI277 and R115,777 to kill human mammary carcinoma cells.