Ceramides promote apoptosis for virus-infected lymphoma cells through induction of ceramide synthases and viral lytic gene expression

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for several human cancers including primary effusion lymphoma (PEL), a rapidly progressive malignancy arising preferentially in immunocompromised patients. With conventional chemotherapy, PEL continues to portend high mortality, dictating the development of novel therapeutic strategies. Sphingosine kinase 2 (SphK2) represents a key gatekeeper for sphingolipid metabolism, responsible for conversion of ceramides to sphingosine-1-phosphate (S1P). We have previously demonstrated that targeting SphK2 using a novel selective inhibitor, ABC294640, leads to intracellular accumulation of ceramides and induces apoptosis for KSHV-infected PEL cells, while suppressing tumor progression in vivo. In the current study, we sought to determine whether specific ceramide/dh-ceramide species and related ceramide synthases (CerS) impact viability for KSHV-infected PEL cells during targeting of SphK2. We found that several specific ceramide and dihydro(dh)-ceramide species and their associated CerS reduce PEL survival and tumor expansion in vitro and in vivo. Moreover, we found that dhC16-Cer induces PEL apoptosis in part through activation of KSHV lytic gene expression. These data further implicate bioactive sphingolipids in regulation of PEL survival, and provide justification for future studies evaluating clinically relevant ceramide analogs or mimetics for their potential as therapeutic agents for PEL.

Keywords: KSHV; ceramide; primary effusion lymphoma; sphingosine kinase.

Conflict of interest statement

CONFLICTS OF INTEREST

All the authors declare no conflict of interest.

Figures

Figure 1. Accumulation of ceramides following targeting of SphK2 within PEL cells

A. The core pathways of sphingolipid metabolism. B.–C. BCBL-1 cells were incubated with the indicated concentrations of ABC294640 (ABC) or vehicle for 16 h, then ceramide and dihydro (dh)-ceramide species were quantified as described in Methods. D. NOD/SCID mice were injected i.p. with 107 BCBL-1 cells. Beginning 21 days later, mice were administered 100 mg/kg ABC or vehicle (n = 10 per group) i.p. once daily, five days per week, for another 21 days. Live PEL cell lysates were recovered from ascites fractions from each of 3 representative vehicle- or drug-treated mice, and intracellular ceramide and dh-ceramide species quantified as above. Error bars represent the S.E.M. for 2 independent experiments, * = p < 0.05; ** = P < 0.01. E.–F. Relative proportions of specific ceramide and dh-ceramide species within vehicle- or drug-treated PEL cells from in vitroE. and in vivoF. experiments are presented.

Figure 2. Targeting SphK2 induces upregulation of ceramide synthases within PEL cells

A.–B. BCBL-1 cells were incubated with the indicated concentrations of ABC or vehicle for 16 h, then transcript A. and protein B. expression of ceramide synthases (CerS1-CerS6) quantified using qRT-PCR and immunoblots, respectively. C. CerS transcripts were quantified using RNA from PEL cells recovered from ascites fractions from each of 3 representative vehicle- or drug-treated mice. Error bars represent the S.E.M. for 2 independent experiments, * = p < 0.05; ** = P < 0.01. D. Spleens from representative vehicle- or drug-treated mice were prepared for routine hematoxylin and eosin (H&E) staining as described in Methods for identification of infiltrating PEL tumors (short arrows), and immunohistochemistry (IHC) was used for identifying CerS2 expression (upper panels, 200x; lower panels, 400x).

Figure 3. C18-Cer and dhC16-Cer induce accumulation of ceramides and apoptosis for PEL cells

A.–C. BCBL-1 cells were incubated with the indicated concentrations of C18-Cer, dhC16-Cer or vehicle for 24 h, then apoptosis A.–B. and protein expression C. quantified as in Methods. D. BCBL-1 cells were incubated with C18-Cer (40 μM), dhC16-Cer (40 μM) or vehicle for 24 h, then intracellular ceramide and dh-ceramide species were quantified as described in Methods. Error bars represent the S.E.M. for 3 independent experiments, * = p < 0.05; ** = P < 0.01.

Figure 4. C18-Cer and dhC16-Cer induce expression of ceramide synthases within PEL cells

A.–B. BCBL-1 cells were incubated with C18-Cer (40 μM), dhC16-Cer (40 μM) or vehicle for 24 h, then transcript A. and protein B. expression of CerS isoforms quantified using qRT-PCR and immunoblots, respectively. C.–D. Cells were transfected with control non-target siRNA (n-siRNA) or CerS2-siRNA for 48 h, then incubated with C18-Cer (40 μM), dhC16-Cer (40 μM) or vehicle for 24 h. Protein expression was detected by immunoblots, and cell apoptosis quantified as above. Error bars represent the S.E.M. for 3 independent experiments, * = p < 0.05; ** = P < 0.01.

Figure 5. C18-Cer and dhC16-Cer induce KSHV lytic gene expression

A.–C. BCBL-1 cells were incubated with C18-Cer (40 μM), dhC16-Cer (40 μM) or vehicle for 24 h, then qRT-PCR used to quantify representative KSHV latent (ORF73) and lytic transcripts (ORF50, ORF74, K8.1, ORF57). Expression of the viral lytic protein K8.1 was determined using immunoblots and IFA. D.–E. BCBL-1 were transfected with control n-siRNA or ORF50-siRNA for 48 h, then incubated with dhC16-Cer (40 μM) or vehicle for 24 h. Viral gene expression and cell apoptosis were quantified by qRT-PCR and flow-cytometry, respectively. F. BCBL-1 were transfected with control n-siRNA or CerS2-siRNA for 48 h, then representative viral transcripts quantified by qRT-PCR. Error bars represent the S.E.M. for 3 independent experiments, * = p < 0.05; ** = P < 0.01.

Figure 6. Exogenous dhC16-Cer suppresses PEL tumor progression in vivo

A.–C. NOD/SCID mice were injected i.p. with 107 BCBL-1 cells. Beginning 24 h later, 20 mg/kg dhC16-Cer or vehicle (n = 10 per group) were administered i.p. 3x/week, for each of 2 independent experiments. Weights were recorded weekly. Images of representative animals and their respective spleens, as well as ascites fluid volumes, were collected at the conclusion of experiments on day 28. Error bars represent the S.E.M. for 2 independent experiments, ** = p < 0.01. D. Spleens from representative vehicle- or dhC16-Cer-treated mice were prepared for routine H&E staining for identification of infiltrating PEL tumors. E. Immunoblots were used to detect CerS protein expression within splenic lysates from representative 2 vehicle- or dhC16-Cer-treated mice.

Figure 7. Short-chain ceramides induce ceramide accumulation and apoptosis for PEL cells

A.–B. BCBL-1 cells were incubated with the indicated concentrations of C2-Cer, C6-Cer, C8-Cer or vehicle for 24 h, then apoptosis quantified as described previously. C. Intracellular ceramide and dh-ceramide species were quantified as above. Error bars represent the S.E.M. for 2 independent experiments, * = p < 0.05; ** = P < 0.01.

Figure 8. Short-chain ceramides induce expression of ceramide synthases within PEL cells

A.–B. BCBL-1 cells were incubated with C2-Cer (50 μM), C6-Cer (6.25 μM), C8-Cer (12.5 μM) or vehicle for 24 h, then transcript A. and protein B. expression of CerS isoforms quantified using qRT-PCR and immunoblots, respectively. C.–D. Cells were transfected with control n-siRNA, CerS2-siRNA or CerS6-siRNA for 48 h, then incubated with C6-Cer (6.25 μM) or vehicle for 24 h. CerS protein expression and cell apoptosis were determined as above. Error bars represent the S.E.M. for 3 independent experiments, * = p < 0.05; ** = P < 0.01.

Figure 9. C6-Cer suppresses PEL formation and induces regression of established PEL tumors in vivo

A.–C. NOD/SCID mice were injected i.p. with 107 BCBL-1 cells. Beginning 24 h later, 20 mg/kg C6-Cer or vehicle (n = 10 per group) were administered i.p. 3x/week, for each of 2 independent experiments. Weights were recorded weekly. Images of representative animals and their respective spleens, as well as ascites fluid volumes, were collected at the conclusion of experiments on day 28. Error bars represent the S.E.M. for 2 independent experiments, ** = p < 0.01. D. Spleens from representative vehicle- or C6-Cer-treated mice were prepared for routine H&E staining. E. Immunoblots were used to detect CerS protein expression within splenic lysates from representative vehicle- or C6-Cer-treated mice. F.–H. NOD/SCID mice were injected i.p. with 107 BCBL-1 cells. Beginning 28 days later, 20 mg/kg C6-Cer or vehicle (n = 10 per group) were administered i.p. 3x/week, for an additional 21 days for each of 2 independent experiments. Weights were recorded weekly, and images of representative animals and their respective spleens, as well as ascites fluid volumes, were collected at the conclusion of experiments on day 49.