Molecular Cancer Research publication

Spindle Assembly Disruption and Cancer Cell Apoptosis with a CLTC-Binding Compound

Bond MJ, Bleiler M, Harrison LE, Scocchera EW, Nakanishi M, G-Dayanan N, Keshipeddy S, Rosenberg DW, Wright DL, Giardina C. Spindle Assembly Disruption and Cancer Cell Apoptosis with a CLTC-Binding Compound. Mol Cancer Res. 2018 Sep;16(9):1361-1372. doi: 10.1158/1541-7786.MCR-18-0178. Epub 2018 May 16. PMID: 29769406; PMCID: PMC6125173.

Abstract
AK3 compounds are mitotic arrest agents that induce high levels of γH2AX during mitosis and apoptosis following release from arrest. We synthesized a potent AK3 derivative, AK306, that induced arrest and apoptosis of the HCT116 colon cancer cell line with an EC50 of approximately 50 nmol/L. AK306 was active on a broad spectrum of cancer cell lines with total growth inhibition values ranging from approximately 25 nmol/L to 25 μmol/L. Using biotin and BODIPY-linked derivatives of AK306, binding to clathrin heavy chain (CLTC/CHC) was observed, a protein with roles in endocytosis and mitosis. AK306 inhibited mitosis and endocytosis, while disrupting CHC cellular localization. Cells arrested in mitosis by AK306 showed the formation of multiple microtubule-organizing centers consisting of pericentrin, γ-tubulin, and Aurora A foci, without apparent centrosome amplification. Cells released from AK306 arrest were unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed division. Like AK306, CHC siRNA knockdown disrupted spindle formation and activated p53. A short-term (3-day) treatment of tumor-bearing APC-mutant mice with AK306 increased apoptosis in tumors, but not normal mucosa. These findings indicate that targeting the mitotic CHC complex can selectively induce apoptosis and may have therapeutic value.
Implication: Disruption of clathrin with a small-molecule inhibitor, AK306, selectively induces apoptosis in cancer cells by disrupting bipolar spindle formation.

Cancer Prevention Research publication

Regulation of VDR Expression in Apc-Mutant Mice, Human Colon Cancers and Adenomas

Giardina C, Nakanishi M, Khan A, Kuratnik A, Xu W, Brenner B, Rosenberg DW. Regulation of VDR Expression in Apc-Mutant Mice, Human Colon Cancers and Adenomas. Cancer Prev Res (Phila). 2015 May;8(5):387-99. doi: 10.1158/1940-6207.CAPR-14-0371. Epub 2015 Apr 14. PMID: 25873367; PMCID: PMC4417398.

Abstract
One variable that may affect the ability of vitamin D to reduce colon cancer risk is the expression of its high-affinity receptor, VDR. Here, we show that vitamin D does not reduce tumor formation in Apc(Δ14/+) mice and that VDR expression is lost in the majority of the colon tumor cells. The extent of VDR loss corresponded inversely to the level of β-catenin nuclear localization and could be observed in early lesions composed of just a few crypts. Analysis of reported VDR regulators showed that the repressing class I histone deacetylases (HDAC) were significantly elevated in the tumors (up to 4-fold), whereas the VDR-activating retinoid X receptors (RXR) were downregulated (∼50%). Expression of the Slug repressor was also increased, but was found primarily in stromal cells. Analysis of epigenetically active compounds on colon cell lines and intestinal organoids showed that HDAC inhibitors were particularly adept at stimulating VDR expression. Treatment of tumor-bearing Apc(Δ14/+) mice with the HDAC inhibitor panobinostat increased VDR expression in the tumors and normal mucosa. The RXR agonist bexarotene failed to activate VDR expression, indicating that RXR ligands were not limiting. Analysis of human microarray data indicated that VDR mRNA is frequently downregulated in colon adenomas, which correlated positively with RXRA expression and inversely with HDAC 2 and 8 expression. Human adenomas showed variable VDR protein expression levels, both between and within individual lesions. Determining the mechanisms of VDR regulation in colon neoplasms may significantly enhance our ability to use vitamin D as a cancer prevention agent.

Cell Stress and Chaperones publication

Hyperbaric oxygen therapy (HBOT) suppresses biomarkers of cell stress and kidney injury in diabetic mice

Verma R, Chopra A, Giardina C, Sabbisetti V, Smyth JA, Hightower LE, Perdrizet GA. Hyperbaric oxygen therapy (HBOT) suppresses biomarkers of cell stress and kidney injury in diabetic mice. Cell Stress Chaperones. 2015 May;20(3):495-505. doi: 10.1007/s12192-015-0574-3. Epub 2015 Feb 4. PMID: 25648080; PMCID: PMC4406928.

Abstract
The disease burden from diabetic kidney disease is large and growing. Effective therapies are lacking, despite an urgent need. Hyperbaric oxygen therapy (HBOT) activates Nrf2 and cellular antioxidant defenses; therefore, it may be generally useful for treating conditions that feature chronic oxidative tissue damage. Herein, we determined how periodic exposure to oxygen at elevated pressure affected type 2 diabetes mellitus-related changes in the kidneys of db/db mice. Two groups of db/db mice, designated 2.4 ATA and 1.5 ATA, were treated four times per week with 100 % oxygen at either 1.5 or 2.4 ATA (atmospheres absolute) followed by tests to assess kidney damage and function. The sham group of db/db mice and the Hets group of db/+ mice were handled but did not receive HBOT. Several markers of kidney damage were reduced significantly in the HBOT groups including urinary biomarkers neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (CyC) along with significantly lower levels of caspase-3 activity in kidney tissue extracts. Other stress biomarkers also showed trends to improvement in the HBOT groups, including urinary albumin levels. Expressions of the stress response genes NRF2, HMOX1, MT1, and HSPA1A were reduced in the HBOT groups at the end of the experiment, consistent with reduced kidney damage in treated mice. Urinary albumin/creatinine ratio (ACR), a measure of albuminuria, was significantly reduced in the db/db mice receiving HBOT. All of the db/db mouse groups had qualitatively similar changes in renal histopathology. Glycogenated nuclei, not previously reported in db/db mice, were observed in these three experimental groups but not in the control group of nondiabetic mice. Overall, our findings are consistent with therapeutic HBOT alleviating stress and damage in the diabetic kidney through cytoprotective responses. These findings support an emerging paradigm in which tissue oxygenation and cellular defenses effectively limit damage from chronic oxidative stress more effectively than chemical antioxidants.