Antitumor Effects of Progesterone Alone and in Combination with Temozolomide Against Neurogenic Tumors Open Access

Patel, Neil Rajendra (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/n583xv487?locale=en
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Abstract

We investigated the antitumorgenic effects of the hormone progesterone (P4) against neurogenic human tumor cells in vitro. First, natural P4 and medroxyprogesterone acetate (MPA), a synthetic progestin, were tested for their cytotoxic effects in neuroblastoma (SK-N-AS) cells. Cell death was measured and we observed that P4 was unable to elicit significant cell death during a 72-hour single exposure. However, repeated 3- and 6-day exposures of P4 at high concentrations (20, 40, 80 µM) significantly (P<0.05) reduced the viability of SK-N-AS cells. Interestingly, MPA was unable to greatly reduce cell viability regardless of exposure duration. We then investigated the antitumor effects of P4 alone and in combination with Temozolomide (TMZ), a chemotherapeutic agent used to treat glioblastoma multiforme (GBM), the most aggressive of neurogenic cancers. We exposed GBM cells (U87MG) to P4 and/or TMZ either as a single exposure for 72 hours or repeated exposures for 3 and 6 days. Again, single exposures to either drug were unable to induce cell death. At high concentrations (20, 40, 80 µM) with repeated exposure, P4 significantly (P<0.05) reduced the viability of U87MG cells. Repeated TMZ exposures also demonstrated some significant reduction in U87MG cell viability at high concentrations (25, 50, 75, 100, 200 µM). Next, we combined TMZ with P4 to examine whether P4 would enhance the cytotoxic effects of TMZ against U87MG cells following repeated exposures. We used a concurrent drug exposure strategy for 3 and 6 days and observed that P4 enhanced the cytotoxic effects of TMZ in combination as compared to TMZ alone but the combination was still less effective than P4 alone. These findings suggest that P4 alone is a potent anti-tumor agent at high concentrations and enhances the cytotoxic efficacy of TMZ in combination in U87MG cells. Furthermore, a combination of the two drugs was also most effective in reducing GBM cell migration. Finally, we examined the modulatory effect of P4 and TMZ alone or in combination on the EGFR/PI3K/Akt/mTOR signaling pathway. Western blot data suggest that P4 alone or in combination suppresses this signaling in U87MG cells and thereby suppresses cell proliferation as evidenced by reduced expression of proliferative cell nuclear antigen (PCNA).

Table of Contents

Introduction...1
Materials and Methods...7

Cell Culture...7
Experimental Design for in vitro cell death studies...7
GBM cell viability with combinatorial drug treatment...8
MTT assay...8
PI Staining...9
Western blot analysis...10
Cell Migration...10
Statistical Analysis...11

Results...12

[A] Effects of P4 and MPA Treatment on Neuroblastoma Cell Viability

Figure 1: Single Exposure to P4 in SK-N-AS cells...12
Figure 2: Repeated P4 exposure in SK-N-AS cells...13
Figure 3: PI staining of SK-N-AS cells...14
Figure 4: Repeated MPA exposure in SK-N-AS cells...15
Figure 5: MPA vs. natural P4 exposure in SK-N-AS cells...16

[B] Effects of P4 and TMZ treatment on GBM cell viability

Figure 6: Single exposure to P4 in U87MG cells...17
Figure 7: Repeated P4 Exposure in U87MG cells...18
Figure 8: Single Exposure to TMZ in U87MG cells...19
Figure 9: Repeated TMZ Exposure in U87MG cells...20
Figure 10: P4 and TMZ Combined Exposure in U87MG cells...21

[C] Effects of P4 and TMZ treatment on GBM cell migration

Figure 11: U87MG Cell Migration following P4 and TMZ Exposure...22

[D] Effects of P4 and TMZ treatment on GBM protein expression

Figure 12: P4 modulates cell proliferation in GBM through the EGFR/PI3K/Akt/mTOR pathway...23

Discussion...25

Figure 13: Natural Progesterone vs. MPA...27
Figure 14: EGFR/PI3K/Akt/mTOR Signaling Pathway...31

References...36
Disclosures...40

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