Solenopsin A and analogs exhibit ceramide-like biological activity
- Isabella Karlsson1,
- Xin Zhou2,
- Raquela Thomas3,
- Allorie T Smith4,
- Michael Y Bonner1,
- Pooja Bakshi6,
- Ajay K Banga6,
- J Phillip Bowen6,
- Ghassan Qabaja7,
- Shavon L Ford7,
- Matthew D Ballard7,
- Kimberly S Petersen7,
- Xuechen Li5,
- Guangping Chen5,
- Besim Ogretmen3,
- Jin Zhang2,
- E Blake Watkins4,
- Rebecca S Arnold8 and
- Jack L Arbiser1, 9Email author
© Karlsson et al.; licensee BioMed Central. 2015
Received: 17 April 2015
Accepted: 21 April 2015
Published: 8 May 2015
(−)-Solenopsin A is a piperidine alkaloid that is a component of the venom of the fire ant Solenopsis invicta. Previously, we have demonstrated that solenopsin exhibit anti-angiogenic activity and downregulate phosphoinositol-3 kinase (PI3K) in the p53 deficient renal cell carcinoma cell line 786-O. Solenopsin has structural similarities to ceramide, a major endogenous regulator of cell signaling and cancer therapy induced apoptosis.
Different analogs of solenopsin were synthesized in order to explore structure-activity relationships. The anti-proliferative effect of solenopsin and analogs was tested on six different cell lines, including three tumor cell lines, two normal cutaneous cell lines, and one immortalized hyperproliferative cell line. FRET-based reporters were used to study the affect of solenopsin and analogs on Akt activity and PDK1 activation and sucrose density gradient fractionation was performed to examine recruitment of PTEN to membrane rafts. Western-blotting was used to evaluate the affect of solenopsin and analogs on the Akt and the MAPK 44/42 pathways in three different tumor cell lines. Measurement of cellular oxygen consumption rate together with autophagy staining was performed to study mitochondrial function. Finally, the affect of solenopsin and analogs on ROS production was investigated.
In this paper we demonstrate that solenopsin analogs with potent anti-proliferative effects can be synthesized from inexpensive dimethylpyridines. To determine whether solenopsin and analogs act as ceramide analogs, we examined the effect of solenopsin and analogs on two stereotypic sites of ceramide activity, namely at lipid rafts and mitochondria. We found that native solenopsin, (−)-solenopsin A, inhibits functional Akt activity and PDK1 activation in lipid rafts in a similar fashion as ceramide. Both cis and trans analogs of solenopsin reduce mitochondrial oxygen consumption, increase reactive oxygen, and kill tumor cells with elevated levels of Akt phosphorylation. However, only solenopsin induces mitophagy, like ceramide.
The requirements for ceramide induced mitophagy and inhibition of Akt activity and PDK1 activation in lipid rafts are under strict stereochemical control. The naturally occurring (−)-solenopsin A mimic some of the functions of ceramide and may be therapeutically useful in the treatment of hyperproliferative and malignant disorders of the skin, even in the presence of elevated levels of Akt.
KeywordsSolenopsin A Ceramide Akt Mitophagy Reactive oxygen
(+)-Solenopsin A and (−)-solenopsin A were synthesized as HCl salts as previously described . Compound S11-S14 were synthesized by deprotonation of 2,6-dimethylpyridine (S12-S14) or 2,4,6-trimethylpyridine (S11) by n-butyllithium, followed by addition of alkyl bromides (Figure 1). S15 was synthesized by treating pyridine-2-carboxaldehyde with the Grignard reagent decylmagnesium bromide (Figure 1). The solenopsin analogs (S11-S15) were successfully obtained after hydrogenation of the various 2-alkylpyridines (Figure 1). Detailed synthetic procedures and characterization data can be found in the Additional file 1.
Cells and culture conditions
In this study eight different cell lines were used: human A375 melanoma cells, human A2058 melanoma cells, immortalized murine endothelial SVR cells [11-13], primary human melanocytes, primary human keratinocyts, HaCaTs (immortalized human keratinocytes), murine embryonic NIH3T3 fibroblast cells, and human UM-SCC1A squamous carcinoma cells. All cell lines were grown in DMEM with 10% fetal bovine serum, except for primary keratinocytes which were grown in serum free keratinocyte growth media and primary melanocytes that were grown in complete melanocyte growth media
A375, A2058, SVR, primary melanocyte, primary keratinocyte, and HaCaT cells were treated with test compounds for 24 hours, followed by cell counting with a Coulter Counter. All compounds were tested in quadruplicates. For A375s, A2058s, SVRs, and primary melanocytes 50,000 cells/well were plated. Due to difficulty growing the cells, primary keratinocytes and HaCaTs were plated at a concentration of 20,000 cells/well, and 15,000 cells/well respectively. All cells were treated with 20 μM of ceramide C2. A375s, A2058s, and SVRs were treated with 10 μM of (−)-solenopsin A, (+)-solenopsin A, or analogs S11-S15, whereas primary melanocytes, primary keratinocytes, and HaCaTs were treated with 20 μM of solenopsin and analogs.
FRET-based reporter construct
The development of the biocensors Lyn-PARE and AktAR has been described previously [14,15]. Briefly, AktAR was generated by a fluorescent protein pair, cerulean and cpVE172, sandwiching a forkhead-associated binding domain (FHA1) and an Akt substrate domain (FOXO) . Lyn-PARE was generated by sandwiching full-length PDK1 between a FRET pair, ECFP (cyan fluorescent protein) and citrine (yellow fluorescent protein) , and a motif generated from the Lyn-kinase gene was added to the 5’-end to target the construct to raft microdomains .
Cell transfection and imaging
Cell transfection and imaging was conducted as previously described [14,15]. NIH3T3 cells were treated for 1 h with DMSO solutions of ceramide C2 (50 μM), (+)-solenopsin A (10 and 20 μM), (−)-solenopsin A (10 and 20 μM), and analogs S11-S15 (10 μM). A more detailed description of the experimental procedure can be found in the Additional file 1.
Sucrose density gradient fractionation
Cells were treated for 1 h with 20 μM DMSO solutions of (+)-solenopsin A, (−)-solenopsin A, analogs (S12-S15), or 50 μM of ceramide. Lipid raft fractionation was performed with a 5-40% sucrose discontinuous gradient as previously described [17,18]. After ultracentrifugation, thirteen 385 μL fractions were collected, starting from the top of the tube. Equal volumes of each fraction were analyzed by Western blot with rabbit polyclonal antibodies for caveolin-1 and PTEN. A more detailed description of the experimental procedure can be found in the Additional file 1.
Western blot analysis
Cells were grown in T-25 flasks until 80% confluent followed by treatment for 24 h with 10 μM DMSO solutions of (+)-solenopsin A, (−)-solenopsin A, or analogs (S11-S15). Sample aliquots normalized for protein quantities were size fractionated by 10% SDS-PAGE, and the proteins were transferred to a PVDF membrane. The blots were incubated in blocking solution; TBS with 5% (wt/vol) powdered nonfat milk for 1 h at room temperature, followed by incubation over night with rabbit polyclonal p-Akt S473, p-MAPK 44/42, and Β-actin.
Measurement of oxygen consumption rate (OCR)
UM-SCC1A cells were plated 15,000 cells/well in 200 μl DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in each well of a 96-well Seahorse plate and incubated overnight at 37°C with 5% CO2. Cells were treated with 10 μM of compound or DMSO and incubated for 24 hours. OCR was measured as pmoles O2/minute using the Seahorse Biosciences instrument per manufacturer’s instructions. Protein amounts in each well were quantified using the Thermo Scientific Pierce Protein Assay, per manufacturer’s instructions.
UM-SCC1A cells were treated with DMSO (control) or 10 μM of (−)-solenopsin A or analog S14 for 18 h. The cells were stained using a Cyto-ID Autophagy Staining assay from Life Technologies.
Measurement of ROS with dihydroethidium (DHE)
Briefly, A375 and SVR cells were treated for 24 h with 10 μM DMSO solutions of (+)-solenopsin A, (−)-solenopsin A, or analogs (S11-S15). Cells were washed, pelleted, suspended in 10 μM DHE and incubated for 10 min. Thereafter cells were counted using a Becton Dickinson FACScan flow cytometer. Mean values of DHE fluorescence intensity were compared and all samples were repeated in triplicate. A more detailed description of the experimental procedure can be found in the Additional file 1.
Determination of cytotoxicity by MTT assay
MTT reagent was added to the tissue inserts after 72 hours treatment with 10 μM of Solenopsin A and analogs S12 and S14 followed by incubation for 3 hours at 37°C with 5% CO2. Thereafter, MTT was extracted from the tissues and the absorbance was measured at 570 nm. Cell viability was calculated using a spreadsheet provided by MatTek; viability of less than 50% was determined to be irritant and cytotoxic. H&E staining of the tissues from the cultures inserts were also performed; see Additional file 1 for more detailed experimental procedures.
Structure-activity relationships of solenopsin and analogs
The effect of ceramide, solenopsin A, and analogs S11-S15 was also assessed on two normal cutaneous cell lines, namely primary melanocytes and primary keratinocytes (Additional file 1: Figure S1). In addition, the activity of the compounds was assessed in HaCaTs, which are immortalized hyperproliferative human keratinocytes (Additional file 1: Figure S1). The analogs S11 and S13, which were inactive in the tumor cell lines, did not have any activity in these cell lines either. Ceramide only showed activity in primary melanocytes and keratinocytes (Additional file 1: Figure S1), but not in malignant A375s, A2058s, and SVRs, (Figure 2). Interestingly, HaCat cells, which represent premalignant keratinocytes, are resistant to ceramide, supporting our hypothesis that loss of response to ceramide may represent an early event in skin carcinogenesis. Solenopsin A and the analogs S12, S14, and S15 had significant activity in all cell lines, including malignant and primary cell lines (Figure 2 and Additional file 1: Figure S1). Solenopsin A and active analogs were shown to be non-toxic to reconstituted skin equivalents (Additional file 1: Figure S2). Normal keratinization was preserved as assessed by routine histology (data not show).
Solenopsin inhibits functional Akt activity and PDK1 activation
Translocation of PTEN to membrane rafts
To examine whether solenopsin recruits PTEN to membrane rafts, as ceramide does, sucrose density gradient fractionation was performed followed by evaluation of the PTEN levels in each fraction. PTEN negatively regulates the PI3K/Akt signaling, which takes place in the lipid raft regions, by converting PIP3 (phosphatidylinostol (3,4,5)-triphosphate) to PIP2 (phosphatidylinositol (4,5)-biphosphate). As most PTEN is usually localized to nonraft regions [14,20,21], studies suggest that ceramide inhibits PI3K/Akt signaling by translocating PTEN from nonraft regions into lipid rafts [15,22,23].
As expected, the amounts of PTEN seem to be higher in lipid raft fractions from cells treated with ceramide (fraction 1–4) (Additional file 1: Figure S3). The compound that appeared to have the largest amount of PTEN in the raft fractions was (−)-solenopsin A (Additional file 1: Figure S3). (+)-solenopsin A and S12-treated cells showed similar amounts of PTEN in the raft fractions as the ceramide treated cells (Additional file 1: Figure S3). The rest of the analogs (S13-S15) appeared to have similar or even lower amounts of PTEN in lipid rafts than the control (Additional file 1: Figure S3). It is worth pointing out that for these experiments the use of a loading control is not possible. Therefore, these results should only be regarded as an indication of the compounds ability to recruit PTEN to membrane rafts. However, the FRET-based analysis was largely consistent with the PTEN localization as demonstrated by sucrose density fractionation.
Solenopsin A and analogs effect on signaling pathways is context dependent
A375 (human melanoma), SVR (murine angiosarcoma), and A2058 (human melanoma) cells treated with solenopsin A and analogs were evaluated by Western-blotting with p-Akt S473, p-MAPK 44/42, and B-actin (Additional file 1: Figure S4 and Additional file 1: Table S1). In A375 human melanoma cells, an up-regulation of p-Akt S473 and p-MAPK 44/42 could be seen for (+)- and (−)-solenopsin A, as well as for analogs S12-S15. In SVR murine angiosarcoma cells on the other hand, p-Akt S473 and p-MAPK 44/42 were down-regulated in all treatment groups, and especially in cells treated with analog S15. The results for the human melanoma A2058 cells were similar to A375 cells but not as pronounced, i.e. there is a slight up-regulation of p-Akt S473 and p-MAPK 44/42 in some of the treatment groups compared to the control. Both human melanoma cell lines (A375 and A2058) have intact p53 and loss of p16ink4a , whereas the murine angiosarcoma cell line (SVR) has defective p53 function due to the presence of SV40 large T antigen . This may account for the observed difference in cell signaling between these cell lines.
Solenopsin A and analogs affect mitochondrial function
Solenopsin A and analogs elevate ROS levels
Ceramides play a role in physiologic cell death, as they are involved in removal of undesired cells, and thus limit both inflammation and neoplasia [28,29]. Ceramides have also been implicated in mediating cell death due to chemotherapy and radiation, and inability to generate ceramides is linked to resistance to these treatments [7,28-30]. Thus, restoration of ceramide is a potential anti-angiogenic and anti-inflammatory modality, but is complicated by difficult synthesis, low stability and rapid metabolism. Therefore, analogs that do not suffer from these disadvantages could be therapeutically beneficial. We noted the similarity of solenopsin to ceramide and hypothesized that solenopsin and analogs might act as ceramide-like agonists. We thus evaluated their ceramide-like activity at both the lipid membrane and mitochondria.
A major obstacle to the widespread use of solenopsin is obtaining sufficient quantities for preclinical and clinical studies. Extraction of solenopsin from ants is not feasible, and therefore large scale synthesis is required. All current synthetic methods suffer from reliance on expensive reagents and multiple steps [31-33]. In this paper, we demonstrate that solenopsin analogs can be synthesized by lithiation of inexpensive industrial dimethylpyridines, followed by alkylation of the lithiated pyridines with alkyl halides, which can be varied. Finally, the alkylated pyridine is hydrogenated to give the solenopsin analogs (Figure 1). Each of these steps is amenable to scale up for industrial production [32,34]. In a previous study by our group we showed that solenopsin analogs with shorter aliphatic side chains lacked anti-proliferative activity in murine angiosarcoma SVR cells . Here we demonstrate that solenopsin analogs with 8 carbon longer side chains (S11 and S13) also lack significant anti-proliferative activity in murine SVR angiosarcoma and human melanoma (A375 and A2058) cells. On the other hand, S14, which has a 4 carbon longer side chain, and S15, which has the same length side-chain but contains a hydroxyl group and lacks the methyl group, are equally as potent as (−)-solenopsin A in all three cell lines. Thus, optimal length of the aliphatic side chain appears to be associated with high anti-proliferative activity.
In this work we show that the naturally occurring (−)-solenopsin A exhibit ceramide-like biological activity in human melanoma cell lines and could therefore be useful in treating hyperproliferative skin disorders. First, we demonstrate that native (−)-solenopsin A mimics canonical functions of ceramide on cells, namely inhibition of Akt activity and PDK1 activation in lipid rafts as well as induction of mitophagy (Figure 6). Differences in cis-trans geometry and chain length abrogate these functions. Second, the response to solenopsin differs in cells of various genetic backgrounds, with no effect or elevation of Akt phosphorylation in cells with wild type p53 (A375, A2058), while decreasing Akt phosphorylation in cells with defective p53 (SVR, 786-O). Solenopsin and analogs are also effective in killing cells with elevated Akt phosphorylation, which is an adverse prognostic factor in most cancers. Based upon the novel hypothesis that restoration of ceramide-like signaling may be beneficial in the treatment of neoplastic and hyperproliferative skin disorders, our assessment of solenopsin and analogs on normal cutaneous cells and premalignant cells demonstrate the potential of solenopsin and analogs to treat hyperproliferative disorders when compared to ceramide.
The data set supporting the results of this article is included within the article and its additional file: Supplementary Material Solenopsin.
Forkhead box protein
Oxygen consumption rate
- PIP2 :
- PIP3 :
Phosphatase and tensin homolog
This work was funded by Dr. Arbiser's NIH R01 AR047901, the Margolis Foundation, Rabinowitch-Davis Foundations and Dr. Zhang's NIH R01 DK073368.
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