Dihydroceramide desaturase knockdown impacts sphingolipids and apoptosis after photodamage in human head and neck squamous carcinoma cells

Abstract

Background: Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis.

Aim: Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4.

Materials and methods: Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed.

Results: Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced.

Conclusion: Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.

Figure 1

The de novo sphingolipid biosynthesis pathway.

Figure 2

Dihydroceramide desaturase 1 (DES) is down-regulated by DES knockdown. UM-SCC-22A cells were transfected with siRNA targeted against non-targeted control (siControl; 25 nM) or DES (siDES; 25 nM). Twenty-four hours after transfection, cells were collected, seeded in fresh growth medium, incubated at 37°C for an additional 24 h, collected, and processed for real-time-polymerase chain reaction (PCR) (A) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/western immunoblotting (B and C). A: DES mRNA levels are expressed in arbitrary units. The data are shown as the mean±SEM, *p≤0.05, n=5-6. B: DES knockdown was confirmed in two independent experiments. C: Following overnight exposure to Pc 4 (250 and 500 nM), cells were irradiated with red light, incubated at 37°C for 2 h, collected and processed for SDS-PAGE/western immunoblotting. B and C: Equal protein loading was verified using anti-heat shock protein 90 (HSP-90).

Figure 3

Dihydroceramide desaturase 1 (DES) knockdown suppressed caspase 3-like (DEVDase) activation after photodynamic therapy (PDT). Following 2-h incubations post-PDT, cells were collected, lysed, and DEVDase activity was measured using Ac-DEVD-AMC as the fluorogenic substrate. The data are expressed as ratios of PDT-treated versus untreated controls and are shown as the mean±SEM, n=3-7. *DES knockdown suppresses PDT-induced DEVDase activation (p≤0.05).

Figure 4

Dihydroceramide desaturase 1 (DES) knockdown suppressed mitochondrial depolarization, apoptosis and cell death after photodynamic therapy (PDT). Following 24-h incubation post-PDT, cells were collected and processed for flow cytometry (A, B), or stained with trypan blue and counted (C). JC-1 and annexin V/propidium iodide (PI) staining were used to detect mitochondrial membrane potential (A) and apoptosis (B), respectively. The data are shown as the mean±SEM, n=3-7. The significance (p≤0.05) is indicated as follows: *DES knockdown affects PDT-induced, mitochondrial depolarization, apoptosis, or cell death; +apoptosis is different between the two PDT doses.