Date of Award

2008

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Kelley Kiningham

Second Advisor

Gary Rankin

Third Advisor

Monica Valentovic

Fourth Advisor

Michael Moore

Fifth Advisor

Lawrence Grover

Abstract

Neuroblastoma is an extra-cranial solid tumor of the nervous system occurring predominantly in infants and children younger than five years of age. Neuroblastoma presents a challenge to therapy primarily due to a decreased responsiveness to anticancer agents like cisplatin (CDDP), leading to recurrence. Vitamin A and its derivatives, known as retinoids, are preventive against cancer and induce differentiation in some cell lines. Retinoids are now being tested clinically for the treatment of neuroblastoma. One major limitation to retinoid therapy is the development of chemoresistance. In the present study human SK-N-SH neuroblastoma cells pretreated with 10 μM all-trans retinoic acid (ATRA) for 24 h followed by CDDP (0.5 - 25 μM), exhibited a decrease in caspase-3 proteolysis and activity (24, 48, 72 and 96 h posttreatment).

In order to elucidate the mechanism of retinoid-mediated resistance we analyzed levels of proteins regulated by nuclear factor kappa binding protein (NFκB) involved in intrinsic apoptosis. Prior exposure to ATRA for 24 h enhanced bcl-xL and MnSOD levels in the CDDP treated SK-N-SH cells which could contribute to chemoresistance. Furthermore, ATRA pretreatment attenuated the CDDP-dependent increase in bax, implicating a reduction in the proapoptotic protein to play a role in the observed drug resistance. However, experiments using the SN50 inhibitor peptide suggested that NFκB does not contribute to ATRA pretreatment-induced chemoresistance. This conclusion further indicates that factors other than NFκB mediate the ATRA dependent modulation of the intrinsic apoptotic process.

Continuing our investigation we observed that superoxide dismutase two (SOD2) siRNA attenuated CDDP-dependent caspase-3 activity and cleavage, having no effect on retinoid mediated chemoresistance under the conditions of the experiment. Similarly, we found that the dominant negative against the retinoic acid receptor alpha (RARα) produced no change in the retinoid-induced chemoresistance in the SK-N-SH cells. In addition, we demonstrated that exposure to ATRA significantly increased phosphorylation of the AKT protein at 30 min, 1h, 4 h, 24 h and 48 h time points. We concluded that although MnSOD and RARα did not contribute to retinoid-induced chemoresistance under the experimental conditions, activation of AKT may contribute, at least in part, to the phenomenon.

The chemotherapeutic agent CDDP is frequently used to treat advanced stages of many human cancers including neuroblastoma. The beneficial effect of CDDP in treating malignancies is hypothesized to involve oxidative stress-induced apoptosis. Oxidative stress also produces adaptive responses by altering the function and expression of several proteins including the superoxide dismutase (SOD) enzymes. However, little is known about the role of the endogenous SOD enzymes in neuroblastoma chemoresistance to CDDP. In the current study CDDP (5-25 µM) significantly increased MnSOD activity and mRNA independent of concentration and NFκB activity in SK-N-SH cells. In contrast, CDDP decreased Cu/ZnSOD expression without altering the activity. Suppressing MnSOD gene expression using siRNA technology resulted in a significant decrease in CDDP-dependent caspase-3 activation and cleavage suggesting that MnSOD in part, potentiated apoptosis. The ability of MnSOD to contribute to CDDP-induced apoptosis was hypothesized to result from the prooxidant effects of hydrogen peroxide produced by the dismutation reaction. In support of this hypothesis, prior incubation of the SK-N-SH cells with the thiol antioxidant, N-acetyl-L-cysteine (NAC), reduced CDDP-mediated increases in caspase- 3 activity.

Following the preceding studies, we established an increase in MnSOD expression and an acute ATRA pretreatment dependent chemoresistance in the SHSY-5Y cells consistent with that observed in the SK-N-SH neuroblastoma line. The findings of the present report established for the first time that ATRA induces chemoresistance within 24 h of prior exposure to the retinoid. This finding suggests that caution should be taken while developing therapeutic strategies using ATRA as a therapeutic tool in neuroblastoma. The present report also provides data elaborating on the possible mechanism by which MnSOD contributes to CDDP-induced apoptosis in neuroblastoma, thereby allowing the development of better therapeutic regimens to treat the disease.

Subject(s)

Neuroblastoma.

Pharmacology.

Retinoids.

Vitamin A.

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