Date of Award

7-11-2019

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Christine Metz, PhD

Abstract

Cisplatin is a widely employed chemotherapeutic agent used in the treatment of numerous cancers, including colon cancer. Approximately 30% of all cancer patients treated with cisplatin experience acute kidney injury (AKI). Approximately 90% of patients treated with cisplatin also have urinary magnesium (Mg) losses causing hypomagnesemia. Mg losses are proposed to further aggravate AKI. Currently, there are no methods to successfully treat or prevent cisplatin-induced AKI. Mg is the most abundant intracellular divalent cation in living organisms having a wide variety of functions. It plays important roles in genomic stability, maintenance of cellular anti-oxidant status, regulation of cell cycle progression, cellular energetics, immuno-inflammatory reactions and apoptosis. It acts as an essential co-factor in more than 300 enzymatic reactions in the body. Mg and calcium (Ca) functions and metabolism are related. Mg serves as nature’s physiological Ca channel antagonist. Ca is a remarkably multi-functional signaling ion and directs the replication, development/ differentiation, function and eventual death of cells. Ca is the most important second messenger in living systems and its intracellular concentration is tightly regulated. There are several ATP dependent pumps employed for this purpose, as well as other mechanisms such as physico-chemical properties of other elements, such as Mg, which act as gates for intracellular Ca entry. If the intracellular Ca concentration increases in an unregulated manner, various signaling pathways are activated leading to the acquisition of pathological characteristics. Several epidemiological studies have linked Mg deficiency and increased Ca:Mg ratios with a higher incidence of colon cancer and mortality. Less than 50% of the US population consumes the Recommended Dietary Allowance (RDA) for Mg and few supplements provide more than 20% of the RDA for Mg. By contrast, oral Ca supplementation is prevalent, especially among the elderly population. Thus, the biological effects of the studies described in this thesis may have potential widespread clinical implications.

The purpose of this thesis is two-fold, the first objective examines the effects of Mg deficiency on cisplatin-induced AKI and tumor growth in a mouse model of colon cancer. The second objective examines the effect of Mg deficiency in the setting of normal Ca levels (i.e. higher Ca:Mg ratios) on the mechanism of migration of colon cancer cells, as well as the degree of aneuploidy (a marker of genomic instability) in these cells.

Based on the above findings and our objectives, we hypothesized that: 

  1. Magnesium deficiency (MgD) will increase cisplatin-induced acute kidney injury in CT26 tumor-bearing BALB/c mice and Mg supplementation (MgS) will protect against this effect without impairing cisplatin-mediated tumor killing.
  2. MgD (with higher Ca:Mg ratios) will enhance CT26 colon cancer cell motility in vitro and genomic instability by modulating intracellular Ca levels and Calpain signaling pathways and promoting oxidative stress.

Using a CT26 colon tumor-bearing mouse model where cisplatin alone (20mg/kg cumulative dose) produced minimal kidney injury, Mg deficiency significantly worsened cisplatin-induced AKI, as determined by biochemical markers (blood urea nitrogen and plasma creatinine) and histological renal changes, as well as markers of renal oxidative stress, inflammation, and apoptosis. By contrast, Mg supplementation (MgS) blocked cisplatin-induced kidney injury. MgD was associated with significantly larger CT26 tumors in BALB/c mice when compared to normal-fed control mice and MgS significantly reduced cisplatin-mediated tumor killing in vivo. Finally, MgS did not compromise cisplatin’s anti-tumor efficacy in vivo.

Next, we used the CT26 colon cancer cells to study the interplay of Ca and Mg and to study the effect of MgD (with varying Ca:Mg ratios) on cell migration and genomic instability in vitro. We observed that MgD-treated cells showed higher intracellular Ca levels with increased TRPM7 expression. MgD also increased oxidative stress in CT26 colon cancer cells. Furthermore, MgD enhanced the motility of the colon cancer cells. This was consistent with the observed degradation of full-length E-cadherin, β-catenin and N-terminal FAK, a cytoplasmic tyrosine kinase, which negatively regulates FAK activity by increasing the cycling of the focal adhesions. MgD also degraded IκBα and the transactivation domain containing C-terminal of NF-κB p65 (RelA). We also observed enhanced degradation of the p53 protein, as well as its transcriptional repression in

MgD-exposed CT26 cells. In addition, CT26 cells grown in MgD media showed evidence of genomic instability as compared to control cells (grown in media containing 100% of the recommended amount of Mg); loss of p53 and associated genomic instability, would be expected to increase metastatic potential. When the Ca:Mg ratio was kept as 1 none of these effects were observed.

The results described in this thesis advance the field because we have shown that MgD exacerbates cisplatin-AKI and MgS protects against it in a colon cancer mouse model. Further, we have shown that MgD leads to larger tumors and compromises the chemotherapeutic effects of cisplatin. We have also shown for the first time that MgD promotes oxidative stress, genomic instability and migration of CT26 colon cancer cells potentially by increasing intracellular Ca. These findings support further studies using various tumor cell lines, mouse models of cancer and clinical studies to better investigate and further elucidate the biology of Mg as it relates to renoprotection following cisplatin treatment (and possibly other nephrotoxic drugs) and cancer cell biology, particularly cell migration and metastasis.

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