Scutellaria baicalensis targets the hypoxia-inducible factor-1α and enhances cisplatin efficacy in ovarian cancer.

Hypoxia-inducible factor-1alpha (HIF-1α) is aberrantly upregulated in tumors and implicated in angiogenesis, metastasis, and drug resistance. Therefore, developing treatments that target HIF-1α may be a viable therapeutic approach. In Traditional Chinese Medicine (TCM), Scutellaria baicalensis (SB) is used for the treatment of cancer but the anti-cancer mechanisms are not known. We examined the effects of SB on HIF-1α expression in ovarian cancer (OC) cell lines grown under normoxic and hypoxic conditions. SB treatment attenuated HIF-1α expression in cancer cell lines. Treatment of cells with cycloheximide (CHX) reduced HIF-1α levels similar to cells treated with SB. Furthermore, SB-induced HIF-1α inhibition was abrogated by the proteasomal inhibitor MG132 and a lysosome inhibitor, chloroquine. Activation of PI3K/AKT and MAPK/ERK seen in OC cells was reduced with SB. Pretreatment of cells with LY294002 (phosphoinositide 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase inhibitor) reduced HIF-1α expression comparable to SB-treated cells. SB potentiated the anti-growth effects of cisplatin on OC cells by attenuating the expression of HIF-1α, ABCG1, and ABCG2. Taken together, the findings suggest that targeting HIF-1α with SB could be an effective treatment strategy for cancer and SB can improve the sensitivity of cancer cells to cisplatin, which is a major challenge in therapy for ovarian tumors.

The Chinese herbs Scutellaria baicalensis and Fritillaria cirrhosa target NFκB to inhibit proliferation of ovarian and endometrial cancer cells.

The herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) are widely used in Chinese medicine to treat several aliments and as an adjuvant to chemotherapy of lung cancer. No information is available regarding the two herbs' influence on ovarian and endometrial cancer. To fill this data gap we compared cell growth responses to SB and FC in ovarian and endometrial cancer cell lines. Dose-dependent cell growth inhibition was observed following higher doses in all cell lines while lower doses stimulated growth in only endometrial cell lines. Higher doses of SB and FC significantly decreased cell growth on soft agar and decreased the invasive potential of cancer cells. Treatment of cells with both herbs resulted in activation of caspase-3, G0 /G1 phase cell cycle arrest, downregulation of cyclins D1 and D3 and induction of p27. Both herbs decreased NFκB DNA binding, reduced expression of phosphorylated IκBα, abrogated NFκB activation, and downregulated NFκB-regulated metastasis-promoting proteins in cancer cells. Furthermore, knockdown of NFκB attenuated SB- and FC-induced cell growth inhibition. These results suggest that inhibition of NFκB activation may be an important mechanism for growth suppression by SB and FC. Data indicate that these herbs may represent a new source of agents for NFκB inhibition in cancer therapy.

Vertical Integration and Reinforcement of Basic Science Concepts in an Engaging Clinically Oriented Physiology Pharmacology Session (COPPS).

Medical educators are finding it challenging to ensure strong basic sciences knowledge is built in the allocated time assigned in the innovative medical school curriculum. In this article, we introduce a novel method to vertically integrate basic sciences knowledge during clinical training.

Systemic administration of antisense oligonucleotides simultaneously targeting CK2α and α' subunits reduces orthotopic xenograft prostate tumors in mice.

CK2 is a highly conserved, ubiquitous, signal responsive protein serine/threonine kinase. CK2 promotes cell proliferation and suppresses apoptosis, and increased CK2 expression is observed in all cancers examined. We previously reported that direct injection of antisense (AS) CK2α phosphorothioate oligonucleotides (PTO) into xenograft prostate tumors in mice significantly reduced tumor size. Downregulation of CK2α in tumor cells in vivo appeared to result in overexpression of CK2α' protein. This suggested that in cancer cells downregulation of CK2α might be compensated by CK2α' in vivo, prompting us to design a bispecific (bs) AS PTO (bs-AS-CK2) targeting both catalytic subunits. bs-AS-CK2 reduced CK2α and α' protein expression, decreased cell proliferation, and induced apoptosis in cultured cells. Biodistribution studies of administered bs-AS-CK2 oligonucleotide demonstrated its presence in orthotopic prostate xenograft tumors. High dose injections of bs-AS-CK2 resulted in no damage to normal liver or prostate, but induced extensive cell death in tumor tissue. Intraperitoneal treatment with bs-AS-CK2 PTO decreased orthotopic tumor size and downregulated both CK2 mRNA and protein expression. Tumor reduction was accomplished using remarkably low doses and was improved by dividing the dose using a multi-day schedule. Decreased expression of the key signaling pathway proteins NF-κB p65 and AKT was also observed. We propose that the molecular downregulation of CK2 through bispecific targeting of the two catalytic subunits may be uniquely useful for therapeutic elimination of tumors.

Protein B23/nucleophosmin/numatrin nuclear dynamics in relation to protein kinase CK2 and apoptotic activity in prostate cells.

Protein B23/nucleophosmin/numatrin (B23) is a key nucleolar/nuclear matrix-associated protein required for cell growth-related functions, such as rRNA synthesis. Protein kinase CK2 (CK2) (formerly casein kinase 2, a protein Ser/Thr kinase signal that is involved in cell growth and cell death) mediates phosphorylation of B23, thereby influencing its functional activity. Here we have delineated the dynamics of B23 and its link to CK2 status in response to altered growth stimuli and induction of apoptosis in cultured prostate cells and in rat prostate cells in vivo. Our studies employing PC-3 and ALVA-41 prostate cancer cells demonstrated colocalization of CK2 and B23 in the nucleus. Further, CK2 and B23 underwent coordinate modulation in the nucleus related to their nucleocytoplasmic shuttling in response to induction of apoptotic activity in cells caused by downregulation of CK2 or by treatment with other apoptosis-inducing agents. These alterations in nuclear association of B23 occurred in the absence of a significant change in the level of cytoplasmic B23. Similar studies in the in vivo model of rat prostate epithelial cells subjected to androgen deprivation (that resulted in loss of nuclear CK2 and induction of apoptosis) demonstrated dynamic modulation of nuclear matrix-associated B23 without a significant change in its cytoplasmic level. These changes were reversed by androgen-mediated growth response in the prostate. Our results suggest that CK2-mediated phosphorylation of B23 is essential for its retention in the nucleus and that coordinated nuclear localization of B23 and CK2 is dynamically regulated in response to altered growth status in the cell.

Medical Engineering Education: Designing a Product Innovation and Technology Session for First-Year Medical Students.

The Carle Illinois College of Medicine created an innovative model for medical education that integrates engineering principles into an active learning curriculum. First-year students were introduced to a medical device in an engaging product innovation and technology session. The goals were to discuss the physiology of oxygen saturation and demonstrate the ability to use observation and research to develop a new product idea. Students hypothetically competed with others to raise money from investors to pursue an efficient medical device and attend the users' needs. Student's feedback reflected a positive impact on their understanding of oximetry measurements and product innovation.

Role of protein kinase CK2 in the regulation of tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis in prostate cancer cells.

Protein kinase CK2 (formerly casein kinase 2 or II) is a ubiquitous and highly conserved protein Ser/Thr kinase that plays diverse roles such as in cell proliferation and apoptosis. With respect to the latter, we originally showed that elevated CK2 could suppress various types of apoptosis in prostate cancer cells; however, the downstream pathways that respond to CK2 for mediating the suppression of apoptosis have not been fully elucidated. Here, we report studies on the role of CK2 in influencing activities associated with tumor necrosis factor-related ligand (TRAIL/Apo2-L)-mediated apoptosis in prostate carcinoma cells. To that end, we show that both androgen-insensitive (PC-3) and androgen-sensitive (ALVA-41) prostate cancer cells are sensitized to TRAIL by chemical inhibition of CK2 using its specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). Furthermore, we have shown that overexpression of CK2alpha using pcDNA6-CK2alpha protected prostatic cancer cells from TRAIL-mediated apoptosis by affecting various activities associated with this process. Thus, overexpression of CK2 resulted in the suppression of TRAIL-induced apoptosis via its effects on the activation of caspases, DNA fragmentation, and downstream cleavage of lamin A. In addition, the overexpression of CK2 blocked the mitochondrial apoptosis machinery engaged by TRAIL. These findings define the important role of CK2 in TRAIL signaling in androgen-sensitive and -insensitive prostatic carcinoma cells. Our data support the potential usefulness of anticancer strategies that may involve the combination of TRAIL and down-regulation of CK2.

Protein kinase CK2 modulates apoptosis induced by resveratrol and epigallocatechin-3-gallate in prostate cancer cells.

Resveratrol and epigallocatechin-3-gallate (EGCG) are important candidates as chemopreventive agents by virtue of their ability to induce apoptosis in cancer cells. Casein kinase 2 (CK2) is a ubiquitous protein ser/thr kinase that plays diverse roles in cell proliferation and apoptosis. We have previously shown that overexpression of CK2 suppresses apoptosis induced by a variety of agents, whereas down-regulation of CK2 sensitizes cells to induction of apoptosis. We therefore investigated whether or not CK2 played a role in resveratrol and EGCG signaling in androgen-sensitive (ALVA-41) and androgen-insensitive (PC-3) prostate cancer cells. Resveratrol- and EGCG-induced apoptosis is associated with a significant down-regulation of CK2 activity and protein expression in both the ALVA-41 and PC-3 cells. Overexpression of CK2alpha protected prostatic cancer cells against resveratrol- and EGCG-induced apoptosis. Relatively low doses (10 mumol/L) of resveratrol and EGCG induced a modest proliferative response in cancer cells that could be switched to cell death by moderate inhibition of CK2. These findings characterize, for the first time, the effects of polyphenolic compounds on CK2 signaling in androgen-sensitive and androgen-insensitive prostatic carcinoma cells and suggest that resveratrol and EGCG may mediate their cellular activity, at least in part, via their targeting of CK2. Further, the data hint at the potential of using these polyphenols alongside CK2 inhibitors in combination chemotherapy.

Intracellular hydrogen peroxide production is an upstream event in apoptosis induced by down-regulation of casein kinase 2 in prostate cancer cells.

We have shown previously that down-regulation of CK2 activity (protein kinase CK2, formerly casein kinase 2) by employing its inhibitors apigenin or 4,5,6,7-tetrabromobenzotriazole promotes apoptosis in prostatic carcinoma cells. In an effort to define the downstream mediators of this action, we show that cell apoptosis observed on down-regulation of CK2 is preceded by intracellular generation of hydrogen hydroxide (H2O2) in various normal and cancer cells. In this regard, both androgen-dependent ALVA-41 and androgen-independent PC-3 cells treated with 80 micromol/L apigenin or 4,5,6,7-tetrabromobenzotriazole or with antisense CK2alpha oligonucleotide or small interfering RNA respond similarly to down-regulation of CK2. Interestingly, whereas chemical inhibitors of CK2 elicited H2O2 production in both cancer and noncancer cells, the antisense CK2alpha-mediated down-regulation of CK2 showed significant H2O2 production in cancer cells but had minimal effect in noncancer cells. The basis of this key difference is unclear at present, but this observation may have implications for the therapeutic potential of antisense CK2 oligonucleotide in cancer therapy. The H2O2 production induced by antisense CK2alpha was associated with robust caspase-3 activity, nuclear factor-kappaB nuclear translocation, cytochrome c release, and subsequent DNA fragmentation in prostate cancer cells (ALVA-41 and PC-3). These findings describe, for the first time, a relationship between CK2 and reactive oxygen species, such that CK2 inhibition leads to production of intracellular H2O2, which may serve as a downstream mediator of apoptosis in cancer cells.

Hydrogen peroxide-mediated cytosolic acidification is a signal for mitochondrial translocation of Bax during drug-induced apoptosis of tumor cells.

Absence of the proapoptotic protein Bax renders tumor cells resistant to drug-induced apoptosis. We have shown that hydrogen peroxide (H(2)O(2))-mediated cytosolic acidification is an effector mechanism during drug-induced apoptosis of tumor cells. Here, we report that Bax is critical in determining the sensitivity of tumor cells to H(2)O(2)-induced apoptosis. More importantly, exposure of colorectal carcinoma (HCT116) and leukemia cells (HL60 and CEM) to H(2)O(2) or its intracellular production during drug-induced apoptosis is a signal for mitochondrial translocation of Bax. Furthermore, we provide evidence that drug-induced H(2)O(2)-mediated Bax translocation in tumor cells is caspase independent but involves cytosolic acidification. Inhibiting cytosolic acidification prevents Bax translocation, and contrarily enforced acidification of the intracellular milieu results in mitochondrial recruitment of Bax, even in the absence of a trigger. These findings provide a novel mechanism for mitochondrial translocation of Bax and directly implicate H(2)O(2)-mediated cytosolic acidification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.

Involvement of reactive oxygen species in apoptosis induced by pharmacological inhibition of protein kinase CK2.

It has been reported that inhibition of protein kinase CK2 (CK2) with antisense oligodeoxynucleotides (ODN) is a potent inducer of apoptosis in cancer cells but not in normal cells. In this regard, the apoptotic-inducing effect is attributed to the catalytic activity of the enzyme, which phosphorylates proapoptotic proteins to inhibit their functions. In this study we investigate the role of intracellular redox status in the proapoptotic activity of CK2 inhibition in human leukemia Cem cells. We provide evidence that inhibition of CK2 activity induces apoptotic cell death as evident by activation of caspase 3, DNA fragmentation, and phosphatidylserine externalization. Inhibition of CK2 resulted in a significant increase in intracellular hydrogen peroxide production, which we show as a critical mediator of apoptosis. To that end, apoptotic hallmarks, like DNA fragmentation and phosphatidylserine externalization, were blocked with the specific hydrogen peroxide scavenger catalase. We also show that inhibition of CK2 reduces cytosolic intracellular superoxide, a precursor of hydrogen peroxide. In summary, decreasing CK2 activity increases intracellular hydrogen peroxide, creating an intracellular environment conducive for death execution. Taken together, these data provide information on novel pathways involved in CK2 biology with implications for effective tools against drug-resistant tumors.

Impact of protein kinase CK2 on inhibitor of apoptosis proteins in prostate cancer cells.

We have previously demonstrated that protein kinase CK2 is a potent suppressor of apoptosis in cells subjected to diverse mediators of apoptosis. The process of apoptosis involves a complex series of molecules localized in various cellular compartments. Among the various proteins that modulate apoptotic activity are inhibitors of apoptosis proteins (IAPs) which are elevated in cancers and have been proposed to block caspase activity. We have examined the impact of CK2 signal on these proteins in prostate cancer cells. Cellular IAPs demonstrate distinct localization and responsiveness to altered CK2 expression or activity in the cytoplasmic and nuclear matrix fractions. Modulation of cellular CK2 by various approaches impacts on cellular IAPs such that inhibition or downregulation of CK2 results in reduction in these proteins. Further, IAPs are also reduced when cells are treated with sub-optimal concentrations of chemical inhibitors of CK2 combined with low or sub-optimal levels of apoptosis-inducing agents (such as etoposide) suggesting that downregulation of CK2 sensitizes cells to induction of apoptosis which may be related to attenuation of IAPs. Decreased IAP protein levels in response to apoptotic agents such as TNFalpha or TRAIL were potently blocked upon forced overexpression of CK2 in cells. Together, our results suggest that one of the modes of CK2-mediated modulation of apoptotic activity is via its impact on cellular IAPs.

CK2 signaling in androgen-dependent and -independent prostate cancer.

Protein serine/threonine kinase casein kinase 2 (CK2) is a key player in cell growth and proliferation but is also a potent suppressor of apoptosis. CK2 has been found to be dysregulated in all the cancers that have been examined, including prostate cancer. Investigations of CK2 signaling in the prostate were originally initiated in this laboratory, and these studies have identified significant functional activities of CK2 in relation to normal prostate growth and to the pathobiology of androgen-dependent and -independent prostate cancer. We present a brief overview of these developments in the context of prostate biology. An important outcome of these studies is the emerging concept that CK2 can be effectively targeted for cancer therapy.

Modulation of death receptor-mediated apoptosis by CK2.

Protein kinase CK2 has long been known to be involved in cell growth and proliferation. Recent work has also implicated its role in the suppression of apoptosis. We originally documented that removal of survival or growth stimuli resulted in rapid loss of CK2 from the nuclear matrix and chromatin which preceded induction of apoptosis. Further, we demonstrated that overexpression of CK2 in cells promotes suppression of drug-mediated apoptosis. In the present work, we have extended these observations to demonstrate that CK2 can influence apoptosis mediated via the death receptors. Overexpression of CK2 resulted in suppression of apoptosis mediated by TNF-alpha, TRAIL, and Fas-L in cells responsive to these ligands, whereas downregulation of CK2 resulted in augmentation of apoptosis mediated by these ligands. To our knowledge, this is the first report to show that receptor-mediated apoptosis can be modulated by changes in CK2 in prostate cancer cells. Based on our previous observations together with the evidence presented here, we propose that CK2 has an impact on the process of apoptosis mediated by diverse type of mechanisms thus playing a global role in regulation of apoptotic activity in cells.

Downregulation of CK2 induces apoptosis in cancer cells--a potential approach to cancer therapy.

We have previously documented that naked antisense CK2alpha ODN can potently induce apoptosis in cancer cells in culture and in mouse xenograft human prostate cancer. The effects of the antisense CK2alpha are related to downregulation of CK2alpha message and rapid loss of the CK2 from the nuclear compartment. Here we demonstrate that downregulation of CK2 elicited by diverse methods leads to inhibition of cell growth and induction of apoptosis. The various approaches to downregulation of CK2 employed were transfection with kinase-inactive plasmid, use of CK2alpha siRNA, use of inhibitors of CK2 activity, and use of antisense CK2alpha ODN packaged in sub-50 nm nanocapsules made from tenascin. In all cases, the downregulation of CK2 is associated with loss in cell survival. We have also described preliminary observations on an approach to targeting CK2 in cancer cells. For this, sub-50 nm tenascin-based nanocapsules bearing the antisense CK2alpha ODN were employed to test that the antisense is delivered to the cancer cells in vivo. The results provide the first preliminary evidence that such an approach may be feasible for targeting CK2 in cancer cells. Together, our results suggest that CK2 is potentially a highly plausible target for cancer therapy.

Targeting CK2 for cancer therapy.

Protein kinase CK2 is a highly ubiquitous and conserved protein serine/threonine kinase that has been found to be involved not only in cell growth and proliferation, but also in suppression of apoptosis. CK2 is capable of dynamic intracellular shuttling in response to a variety of signals. It is localized in both the nucleus and cytoplasm in normal cells, but is particularly predominant in the nuclear compartment in cancer cells. CK2 has been found to be uniformly dysregulated in all the cancers that have been examined. Downregulation of CK2 by chemical or molecular methods promotes apoptosis in cells. We have shown that antisense CK2alpha is particularly potent in inducing apoptosis in cancer cells in culture as well as in xenograft models of cancer such as prostate cancer and squamous cell carcinoma of head and neck. The antisense CK2alpha oligodeoxynucleotide (ODN) mediates tumor cell death in a dose- and time-dependent manner such that at an appropriate concentration of the antisense, a complete resolution of the xenograft tumor is observed. Interestingly, normal and benign cells (in culture as well as in vivo) demonstrate a relative resistance to the antisense CK2alpha ODN treatment, which raises the possibility of a significant therapeutic window for this therapy. Further, novel approaches such as the delivery of antisense CK2alpha ODN encapsulated in sub-50-nm tenascin nanocapsules have become available for its targeting specifically in cancer cells. Our studies minimize generally held concerns regarding suitability of CK2 as a target for cancer therapy and provide the first encouraging results for potential future application of this approach for cancer therapy.