Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines

High interstitial level of ATP and its lysate adenosine in the cancer microenvironment are considered a halo mark of cancer. Adenosine acts as a strong immune suppressor. However, the source of ATP release is unclear. We clarified the release of ATP via volume-regulated anion channels (VRACs) in breast cell lines using an ATP luminescence imaging system. We detected a slowly rising diffuse pattern of ATP release that was only observed in undifferentiated cells, not in differentiated primary cultured cells. This was confirmed by suppression with DCPIB, a blocker of VRACs, and shRNA for LRRC8A, an indispensable subunit of VRACs. We herein demonstrated that the inflammatory mediator sphingosine-1-phosphate (S1P), which exists abundantly in the cancer microenvironment, induced a diffuse pattern of ATP release isovolumetrically. The response was dose-dependent and suppressed by the knock-down of LRRC8A. It was also suppressed by blockers of S1P receptor 1 and 2 (W146 and JTE013, respectively). RTqPCR demonstrated the prominent presence of S1PR1 and S1PR2 mRNAs. We discussed the roles of S1P-induced ATP release in the cancer microenvironment.

Hypo-osmotic Stress Induces ATP Release via Volume-regulated Anion Channels in Undifferentiated Mammary Cells

The high interstitial ATP concentration in the cancer microenvironment is a major source of adenosine, which acts as a strong immune suppressor. However, the source of ATP release has not been elucidated. We measured the ATP release during hypotonic stress using a real-time ATP luminescence imaging system in primary cultured mammary cells and in breast cell lines. In primary cultured cells, ATP was intermittently released with transient-sharp peaks, while in breast cell lines ATP was released with a slowly rising diffuse pattern. The diffuse ATP release pattern was changed to a transient-sharp pattern by cholera toxin treatment and the reverse change was induced by transforming growth factor (TGF) β treatment. DCPIB, an inhibitor of volume-regulated anion channels (VRACs), only suppressed the diffuse pattern. The inflammatory mediator sphingosine-1-phosphate (S1P) induced a diffuse ATP release pattern isovolumetrically. The knockdown of A isoform of leucine-rich repeat-containing protein 8 (LRRC8A), the essential molecular entity of VRACs, using shRNA suppressed the diffuse pattern. These results suggest that abundantly expressed VRACs are a conduit of ATP release in undifferentiated cells, including cancer cells.

Cytotoxicity of Seaweed Compounds, Alone or Combined to Reference Drugs, against Breast Cell Lines Cultured in 2D and 3D

Seaweed bioactive compounds have shown anticancer activities in in vitro and in vivo studies. However, tests remain limited, with conflicting results, and effects in combination with anticancer drugs are even scarcer. Here, the cytotoxic effects of five seaweed compounds (astaxanthin, fucoidan, fucosterol, laminarin, and phloroglucinol) were tested alone and in combination with anticancer drugs (cisplatin—Cis; and doxorubicin—Dox), in breast cell lines (three breast cancer (BC) subtypes and one non-tumoral). The combinations revealed situations where seaweed compounds presented potentiation or inhibition of the drugs’ cytotoxicity, without a specific pattern, varying according to the cell line, concentration used for the combination, and drug. Fucosterol was the most promising compound, since: (i) it alone had the highest cytotoxicity at low concentrations against the BC lines without affecting the non-tumoral line; and (ii) in combination (at non-cytotoxic concentration), it potentiated Dox cytotoxicity in the triple-negative BC cell line. Using a comparative approach, monolayer versus 3D cultures, further investigation assessed effects on cell viability and proliferation, morphology, and immunocytochemistry targets. The cytotoxic and antiproliferative effects in monolayer were not observed in 3D, corroborating that cells in 3D culture are more resistant to treatments, and reinforcing the use of more complex models for drug screening and a multi-approach that should include histological and ICC analysis.

R-sulforaphane modulates the expression profile of AhR, ERα, Nrf2, NQO1, and GSTP in human breast cell lines

Our previous study showed remarkable differences in the effect of R-sulforaphane (R-SFN) on the expression of CYPs 19, 1A1, 1A2, and 1B1 in ER(+) MCF7, ER( −) MDA-MB-231, and non-tumorigenic immortalized MCF10A (8). This study aimed to evaluate the effect of R-SFN on phase II enzymes induction and expression of AhR, Nrf2, and ERα in the same breast cell lines. The results showed increased expression of GSTP as a result of treatment with R-SFN in breast cancer cells. An increased NQO1 transcript and protein levels were found in all breast cells, with the most significant increase in MCF7 cells. Similarly, the enhancement of Nrf2 expression was noticed in all tested cells. AhR gene transcript and protein were decreased in MCF7 cells. In MDA-MB-231, increased AhR mRNA was not confirmed at the protein level. No differences were found in the expression of ERα. Overall, the results of the present study extended our earlier suggestions on the possible interference of R-SFN with estrogens homeostasis in breast cancer cells differing in ERα status, as well as in non-tumorigenic immortalized breast epithelial cells. While some of R-SFN effects might be beneficial and useful in breast cancer prevention, the others, particularly GSTP induction, may lead to adverse effects.

Comparative studies of two vegetal extracts from Stokesia laevis and Geranium pratense: polyphenol profile, cytotoxic effect and antiproliferative activity

In this study, two ethanolic extracts, from Stokesia aster (Slae26) and Geranium pratense (Gpre36) respectively, were evaluated in order to assess the cytotoxic activity and potential antiproliferative activity upon the nontumorigenic human epithelial cell line derived from the mammary gland (MCF-12A) and the human breast tumor cell line (BT-20). The selection of the plant species was done on the basis of their chemical composition, specifically combinations of luteolin derivatives with caffeic and gallic acid derivatives. Therefore, the S. laevis ethanolic extract proved its capacity to inhibit the viability of both normal and tumor breast cell lines (i.e., up to 90% cell viability inhibition, IC50 = 42 µg/mL). On the contrary, the G. pratense ethanolic extract proved weak stimulatory effects on the viability of the two human breast cell lines studied. The obtained results were discussed in the contexts of computational studies and drug-likeness bioactivity of seven common luteolin derivatives: luteolin, luteolin-7-O-glucoside/cynaroside, luteolin-5-O-glucoside/galuteolin, luteolin-6-C-glucoside/isoorientin, luteolin-8-C-glucoside/orientin, luteolin-3′,4′-di-O-glucoside and luteolin-7,3′-di-O-glucoside. Computational studies have revealed that the hydrophilic behavior of luteolin derivatives (log P values) does not follow other tested parameters (e.g., polar surface area values), possibly explaining different efficacy concerning the biological properties in vitro. These predictions could be a starting point for studies on the biochemical mechanism by which luteolin derivatives induce biological effects.

Simalikalactone D, a Potential Anticancer Compound from Simarouba tulae, an Endemic Plant of Puerto Rico

Species of the genus Simarouba have been studied because of their antimalarial and antileukemic activities. A group of oxygenated terpenes called quassinoids have been isolated from species of the Simarouba genus, and are responsible for its therapeutic properties. We hypothesized that Simarouba tulae, an endemic plant from Puerto Rico, is a natural source rich in quassinoid compounds with anticancer activity. The leaves were processed and extracted with solvents of different polarities. The extracts were screened for their antiproliferative activity, and it was shown that the chloroform extract was the most active extract. This extract was purified using different chromatographic techniques to afford the quassinoid simalikalactone D (SKD). This compound was further characterized using NMR and X-ray diffraction analysis. A reassessment of original structural assignments for SKD is proposed. SKD showed high cytotoxicity activity, with an IC50 of 55, 58, and 65 nM in A2780CP20 (ovarian), MDA-MB-435 (breast), and MDA-MB-231 (breast) cell lines, respectively. Exposure to SKD led to 15% inhibition of the migration of MDA-MB-231 cells.