Liposomal C6 Ceramide Activates Protein Phosphatase 1 to Inhibit Melanoma Cells

Melanoma is one common skin cancer. In the present study, the potential anti-melanoma activity by a liposomal C6 ceramide was tested in vitro. We showed that the liposomal C6 (ceramide) was cytotoxic and anti-proliferative against a panel of human melanoma cell lines (SK-Mel2, WM-266.4 and A-375 and WM-115). In addition, liposomal C6 induced caspase-dependent apoptotic death in the melanoma cells. Reversely, its cytotoxicity was attenuated by several caspase inhibitors. Intriguingly, liposomal C6 was non-cytotoxic to B10BR mouse melanocytes and primary human melanocytes. Molecularly, liposomal C6 activated protein phosphatase 1 (PP1) to inactivate Akt-mammalian target of rapamycin (mTOR) signaling in melanoma cells. On the other hand, PP1 shRNA knockdown or exogenous expression of constitutively activate Akt1 (CA-Akt1) restored Akt-mTOR activation and significantly attenuated liposomal C6-mediated cytotoxicity and apoptosis in melanoma cells. Our results suggest that liposomal C6 activates PP1 to inhibit melanoma cells.


Introduction
Melanoma is one common skin cancer [1,2,3,4,5]. It is characterized by rapid disease progression and early invasion/metastasis to other organs [6]. It is estimated that metastatic or recurrent melanoma causes over 8000 deaths each year [5]. In addition, melanoma is resistant to almost all traditional chemotherapy agents [1,2,3,4]. Currently, dacarbazine and temozolomide (TMZ) are routinely prescribed for melanoma chemotherapy. Yet, the response rate is often less 15-20% [1,2,3,4]. Therefore, it is urgent to explore novel and more potent anti-melanoma agents.
Ceramides are a family of lipid molecules that are enriched within cell membranes [7,8]. Ceramides could also function as active signaling molecules [7,8]. Among all the ceramides, the short-chain cell permeable ceramides (C2, C4, C6 and C8) have displayed promising antitumor activity, either alone or in combination with traditional anti-cancer agents (reviewed in [9,10,11,12]). C6 ceramide has been tested in melanoma cells, and showed decent in vitro cytotoxicity [13]. Yet, the systematic use of the short-chain ceramides is extremely limited due to their poor solubility [14]. Therefore, liposome-based nanotechnology delivery systems have been developed to assist ceramide delivery in vivo [14,15,16,17,18]. In the current study, we investigated the potential anti-melanoma activity by a liposomal C6 ceramide [14,18]. The underlying mechanisms were also analyzed.

Chemicals and reagents
Liposomal C6 (ceramide), liposome ghost vehicle and free C6 (ceramide) were provided by Bo Zhang's Lab at Tianjin Medical University [19]. The caspase-3 specific inhibitor Ac-DEVD-CHO, the caspae-9 specific inhibitor Ac-LEHD-CHO and the pan caspase inhibitor Ac-VAD-CHO were purchased from Peptide Institute (Osaka, Japan). Antibodies of PP1α/β/λ were obtained from Santa Cruz Biotech (Santa Cruz, CA). All other antibodies utilized in the study were purchased form Cell Signaling Tech (Denver, MA). Cell culture reagents were provided by Calbiochem (Shanghai, China).

Single-stranded DNA (ssDNA) ELISA assay of apoptosis
In the process of apoptosis, DNA denature is a characteristic marker. In the present study, denatured ssDNA was detected via a nucleosomal monoclonal antibody in an ELISA format. Detailed protocol was described in other studies [19,21,22,23]. Briefly, melanoma cells (2.5 ×10 4 /well) were seeded onto 96-well plates. After applied treatment, cell apoptosis was analyzed via the ssDNA ELISA kit (Chemicon, Shanghai, China) according to the attached protocol. The OD value was utilized as a quantitative indicator of cell apoptosis.

Western blots
Cells were washed and incubated in cell lysis buffer [20]. Protein samples were separated by SDS-PAGE gel and electro-transferred to PVDF membranes (Bio-Rad), followed by incubation with primary antibodies [18]. Protein bands were visualized using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz), and by the enhanced chemiluminescence (ECL) reagents [19]. The x-ray films were scanned, acquired in Adobe Photoshop, and analyzed with NIH Image J software.

Protein phosphatase activity assay
Protein phosphatase activity was determined with the [ 32 P] phosphorylase a protocol as previously described [24]. The assay was performed in a 50-μl aliquot that consisted of 50 mM Tris HCl (pH 7.4), 5 mM caffeine, 0.5 mM EGTA, 0.5 mM EDTA, 50 μM β-mercaptoethanol, and 100 ng of aprotinin (protease inhibitor) with or without 2 μg of protein lysates and 500 pmol [ 32 P] phosphorylase a [24]. The assay was initiated by adding the cell lysates and was incubated at 30°C for 5 min. Incubation was rapidly stopped by addition of 30 μl of 60% TCA and 20 μl of BSA (50 mg/ml). Tubes were held in ice for 10 min and then centrifuged at 12,000 g for 5 min. After centrifugation, 32 P radioactivity was counted in 80 μl of clear supernatant in 7 ml of liquid scintillation fluid. protein phosphatase activity was calculated through the same protocol as described [24]. The protein phosphatase activity of liposomal C6 treatment group was normalized to that of untreated control group.

PP1 shRNA knockdown
The pan PP1 shRNA (sc-43545-SH, Santa Cruz) and scramble control shRNA were purchased from Santa Cruz Biotech (Shanghai, China). The PP1 shRNA sequence was described in the previous study [25]. For shRNA transfection, melanoma cells were seeded at 50% confluence. The shRNA vector was introduced by Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. The stable cells expressing PP1 shRNA were selected by puromycin (2.5 μg/ml) for 2-3 weeks. Western blot assay was always performed to test PP1α expression in stable cells.

Constitutively active-Akt1 (CA-Akt1) transfection and stable cells selection
The constitutively active mutant of Akt1 (CA-Akt1) cDNA sequence was provided by Dr. Teng's group at Jining Medical University [26,27]. CA-Akt1 was inserted into the pSuperpuro-GFP vector and was transfected via Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer's protocol. The stable cells were selected by puromycin (2.5 μg/ml) for 2-3 weeks. Western blot assay was performed to test CA-Akt1 in stable cells.

Statistical analysis
The values in the figures were expressed as the means ± standard deviation (SD). Statistical analysis of the data was performed by ANOVA. Values of p < 0.05 were considered as statistically different.  treatment dose-dependently inhibited the number of WM-115 colonies (Fig 1C), suggesting its anti-proliferative activity. Note that liposomal ghost ("Lipo") showed almost no effect on melanoma cell survival nor proliferation (Fig 1A and 1C).

Liposomal C6 inhibits melanoma cell survival and proliferation
The potential effect of liposomal C6 on other melanoma cell lines was also analyzed. Three other established melanoma cell lines (SK-Mel2, WM-266.4 and A-375) were cultured and treated with liposomal C6 (10 μM). MTT assay results in Fig 1D showed that liposomal C6 was cytotoxic against all these melanoma cell lines. To compare the efficiency between regular free C6 and liposomal C6, WM-115 cells were treated with same concentration (10 μM) of free C6 or liposomal C6, MTT results showed that liposome-packed C6 was significantly more potent than free C6 in suppressing WM-115 cells (Fig 1E). Same results were also obtained in other tested melanoma cell lines (Data not shown). The potential activity of liposomal C6 on normal melanocytes (non-cancerous cells) was tested. MTT results in Fig 1F showed that liposomal C6 (10 μM) failed to inhibit survival of B10BR mouse melanocytes and primary human melanocytes, implying its selective cytotoxicity to cancer cells. Collectively, these results indicate that liposomal C6 exerts cytotoxic and anti-proliferative activity against cultured human melanoma cells.

Liposomal C6 activates apoptosis in melanoma cells
Next, we studied the potential effect of liposomal C6 on cell apoptosis. WM-115 cells were treated with indicated concentration of liposomal C6. Results in Fig 2A and 2B showed that liposomal C6 dose-dependently increased activity of caspse-3 and caspase-9 in WM-115 cells. In addition, liposomal C6 (5-25 μM) significantly increased Annexin V percentage ( Fig 2C) and ssDNA ELISA OD (Fig 2D). All these results indicated apoptosis activation by liposomal C6 in WM-115 cells (Fig 2A-2D). To study the role of apoptosis in liposomal C6-induced melanoma cytotoxicity, three caspase-based apoptosis inhibitors were applied. Results showed that the caspase-3 specific inhibitor Ac-DEVD-CHO, the caspae-9 specific inhibitor Ac-LEHD-CHO and the pan caspase inhibitor Ac-VAD-CHO dramatically inhibited liposomal C6 (10 μM)-induced WM-115 cell viability reduction (Fig 2E). ssDNA apoptosis ELISA results in Fig 2F confirmed significant apoptosis activation in three other melanoma cell lines after liposomal C6 (10 μM) treatment. Once again, liposomal C6 was more potent than free C6 in inducing apoptosis in WM-115 cells (Fig 2G). Notably, ssDNA ELISA assay results in Fig 2H demonstrated that liposomal C6 failed to induce significant apoptosis in B10BR mouse melanocytes and primary human melanocytes. These results against confirmed its selective activity in cancerous cells. Collectively, liposomal C6 induces caspase-dependent apoptotic death in melanoma cells.

Liposomal C6 activates protein phosphatase, and inhibits Akt-mTOR signaling in melanoma cells
Previous studies have shown that short-chain ceramides could activate the protein phosphatase 1 (PP1) [28,29] and de-phosphorylates Akt to exert cytotoxic or anti-proliferative activity [30]. We thus analyzed protein phosphatase activity in liposomal C6-treated melanoma cells using the method described [24]. Results demonstrated that liposomal C6 dose-dependently increased protein phosphatase activity in both WM-115 ( Fig 3A) and A-375 melanoma cells (Fig 3B). As a result, Akt activation was largely inhibited (Fig 3C and 3E). In addition, pP70S6K1, the indicator of mammalian targeted of rapamycin (mTOR) activation, was also inhibited (Fig 3C and  3E). Akt and P70S6K1 phosphorylations in WM-115 and A375 cells were quantified ( Fig  3D and 3F). Since, Akt-mTOR activation plays a vital role in melanoma cell survival and proliferation [31], our results suggest that liposomal C6 activates protein phosphatase to inhibit Akt-mTOR signaling and melanoma cell proliferation.

Discussion
Despite the promising anti-cancer activity by the short-chain ceramides [7,10,32], the process of developing these compounds as active pharmaceutical agents has been hampered due to their insolubility [14]. Therefore, liposome-based nanotechnology delivery systems have been  Stable WM-115 cells expressing the pan PP1 shRNA, constitutively-activate mutant Akt1 ("CA-Akt1"), or empty vector ("pSuper-puro") were treated with or without applied concentration of liposomal C6 ceramide developed to assist ceramide delivery in vivo [19,33,34,35]. It has been shown that system delivery of liposomal C6 could offer rapid tissue distribution without causing apparent toxicities [15]. In addition, liposomal C6 showed a selective response to cancerous cells [15,18,19]. Recent studies have also concluded that experimental mice were well-tolerated to the liposomal C6 systematic administration [18,19]. In the current study, our in vitro studies showed that liposomal C6 (ceramide) exerted potent anti-proliferative and pro-apoptotic activities against a panel of human melanoma cell lines (SK-Mel2, WM-266.4, A-375 and WM-115). Its efficiency was better than free C6 ceramide. Intriguingly, liposomal C6 was non-cytotoxic to B10BR mouse melanocytes and primary human melanocytes.
At the molecular level, we showed that shRNA knockdown of PP1 or introduction of CA-Akt1 alleviated liposomal C6-mediated anti-melanoma activity. These results indicate that PP1-mediated Akt-mTOR inactivation mediated, at least in part, liposomal C6's cytotoxicity in melanoma cells. However, it should be noted that PP1 shRNA or CA-Akt1 didn't completely block liposomal C6' cytotoxicity, indicating that other mechanisms besides the PP1-Akt signaling may also contribute to its actions. As a matter of fact, studies have identified other signaling mechanisms by (liposomal) C6 in various cancer cells, including JNK activation [36], AMP activated protein kinase (AMPK) activation [18,37,38,39,40], growth factor receptor degradation [40] and many others. A recent study by Zhang et al., showed that acute treatment (30 min) of liposomal C6 inhibited melanoma cell migration via phosphorylation of PI3K and PKCz [41]. Reversely, knockdown or pharmacological inhibition of PKCz or PI3K restored cancer cell migration following liposomal C6 treatment [41]. It will be interesting to test these signalings in liposomal C6-treated melanoma cells as well.

Conclusions
Metastatic and recurrent melanoma is still a great challenge to treat [6,42,43]. Stage III melanoma patients are often treated adjuvantly with interferon (IFN)-α, yet its response is far from satisfactory. The metastatic melanoma patients (stage IV) have a median survival of 6-10 months even with current treatments, and the 5-year survival is less than 5% [6,42,43]. Therefore, alternative treatment agents are urgently needed [6,42,43]. Our results show that liposomal C6 potently inhibits melanoma cells in vitro. Therefore, the liposomal C6 could be further studied for possible treatment of melanoma.