The anticancer drug cisplatin induces an intrinsic apoptotic pathway inside the inner ear

Abstract

Background and purpose: Ototoxicity is a known adverse effect of cisplatin (CDDP). Since apoptosis is involved in the development of some pathological conditions associated with the administration of anticancer drugs, we examined, using immunohistochemical and electrophysiological techniques, the apoptotic changes in the cochlea of Sprague-Dawley (SD) rats after an injection of CDDP (5 mgkg(-1) body weight).

Experimental approach: Luciferase assays were used to determine the different caspase activities and ATP levels in protein extracts of whole cochleae. The expression of several apoptotic-related proteins was measured by means of Western blotting. These analyses were performed 2, 7 and 30 days after the CDDP injection. The auditory brain stem response was obtained before and at the different times after the injection of CDDP, before the animals were killed.

Key results: CDDP significantly increased the levels of caspase-3/7 activity and active caspase-3 protein expression and caspase-3 immunofluorescence staining, caspase-9 activity, and Bax protein expression but decreased Bcl-2 protein expression within the rat cochleae. Threshold shifts were significantly elevated 2 days after CDDP treatment.

Conclusions and implications: These findings support the hypothesis that cisplatin-related apoptosis evokes an intrinsic pathway of pro-apoptotic signalling within the rat cochleae. Thus, selective inhibition of the sequence of events involved in the intrinsic apoptotic pathway could provide a strategy to minimize cisplatin-induced ototoxicity.

Figure 1

Threshold shifts of ABR. The ABR threshold shift was significantly elevated only in the animals killed 2 days after cisplatin (CDDP) administration. Animals from the 7-day and 30-day groups showed a non-significant increase in the ABR threshold shift. ABR, auditory brainstem response; CDDP, cis-diamminedichloroplatinum II.

Figure 2

Cisplatin-related apoptosis in rat cochleae. Caspase-3/7 activity (a) and ATP levels (b) were measured in whole cochlea protein extracts of rats killed 2, 7 or 30 days after CDDP injection. Results are expressed as RLUs (relative light units); ^*P⩽0.05 vs control group. CDDP, cis-diamminedichloroplatinum II.

Figure 3

Active caspase-3 expression. (a) A representative western blot of active p-17 caspase-3 protein expression levels in whole cochlea extracts of rats killed at 2, 7 or 30 days after CDDP injection is shown (left). Densitometric analyses of previous western blots are also shown (right). Results are expressed as arbitrary units; ^*P⩽0.05 vs control. (b) Immunostaining of active caspase-3 in the nuclei of inner ear cells from rats 7 days after a single dose of cisplatin (5 mg kg⁻¹). Cryosections from the cochleae of exposed animals were stained (see Methods section). Increased nuclear caspase-3 immunostaining is seen in several cell populations after administration of cisplatin. MC, marginal cells of stria vascularis; RM, Reissner's membrane; OHCs, outer hair cells; IC, interdental cells of the spiral limbus. Magnification × 400. CDDP, cis-diamminedichloroplatinum II.

Figure 4

Caspase-8 and caspase-9 activities. Caspase-8 (a) and caspase-9 (b) activities were measured in rat cochlea protein extracts of rats killed at 2, 7 or 30 days after CDDP injection. Results are expressed as RLUs (relative light units); ^*P⩽0.05 vs control group. CDDP, cis-diamminedichloroplatinum II.

Figure 5

Bax and Bcl-2 protein expressions. Representative western blots of active Bax (a, left) or Bcl-2 (b, left) protein expressions in whole cochlea extracts of rats killed at 2, 7 or 30 days after CDDP injection are shown. Densitometric analyses of previous western blots are also shown (right). Results are expressed as arbitrary units; ^*P⩽0.05 vs controls. CDDP, cis-diamminedichloroplatinum II.

Figure 6

CDDP treatment modulates total SOD activity in rat cochleae. Total SOD activity is measured in whole cochlear extracts of rats killed at 2, 7 or 30 days after CDDP injection. Results are expressed as U ml⁻¹. One unit of activity is defined as the activity of enzyme required to inhibit the production of formazan by 50%; ^*P⩽0.05 vs control group. CDDP, cis-diamminedichloroplatinum II; SOD, superoxide dismutase.