Aline Pinon

Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes.

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time‐period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calcium‐induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2‐M phases. Furthermore, RT‐PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium‐induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

The P2Y2/Src/p38/COX-2 pathway is involved in the resistance to ursolic acid-induced apoptosis in colorectal and prostate cancer cells

One of the hallmarks of cancer is resistance to apoptosis. Elucidating the mechanisms of how cancer cells evade or delay apoptosis should lead to novel therapeutic strategies. Previously, we showed that HT-29 colorectal cancer cells undergoing apoptosis overexpressed cyclooxygenase-2 (COX-2), in a p38 dependent pathway, to delay ursolic acid-induced apoptosis. Here, we focused on elucidating the upstream signaling pathways regulating this resistance mechanism. The role of ATP as an extracellular signaling molecule took a long time to be accepted. In recent years, ATP and its analogs, via the activation of specific purinergic receptors, have been implicated in many biological processes including cell proliferation, differentiation and apoptosis. In the present report, we have demonstrated a novel role involving purinergic receptors and particularly the P2Y(2) receptor in resistance to ursolic acid-induced apoptosis in both colorectal HT-29 and prostate DU145 cancer cells. We found that ursolic acid induced an increase in intracellular ATP and P2Y(2) transcript levels. Upon activation, P2Y(2) activated Src which in turn phosphorylated p38 leading to COX-2 overexpression which induced resistance to apoptosis in both HT-29 and DU145 cells. Furthermore, Ca(2+)-independent PLA(2) (iPLA(2)) and Ca(2+)-dependent secretory PLA(2) (sPLA(2)) were responsible for arachidonic acid release, the substrate of COX-2. Our findings document that apoptosis triggering was dependent on protein kinase C (PKC) activation in both cell lines after ursolic acid treatment.

HT-29 colorectal cancer cells undergoing apoptosis overexpress COX-2 to delay ursolic acid-induced cell death

Colorectal cancer is one of the most common cancer types and the third leading cause of cancer-related death in the western world. Generally, colorectal cancers are resistant to anticancer drugs. Several lines of evidence support a critical role for cyclooxygenase-2 (COX-2) during colorectal tumorigenesis and its role in chemoresistance. In this study, we focused our interest on the role played by COX-2 in apoptosis induced in HT-29 human colorectal cancer cells by ursolic acid (UA), a triterpenoid found in a large variety of plants. We showed that UA-induced apoptosis and that COX-2 was overexpressed only in apoptotic cells. We demonstrated that this overexpression was mediated by the p38 MAP kinase pathway as inhibiting its activation using a p38-specific inhibitor, SB 203580, abrogated COX-2 expression. Inhibiting COX-2 expression either by using a p38-specific inhibitor or COX-2-specific siRNA increased apoptosis. These results demonstrated that COX-2 was involved in a resistance mechanism to UA-induced apoptosis in HT-29 cells. Cells undergoing apoptosis were able to trigger a resistance mechanism by overexpressing a protein such as COX-2 to delay their death. Furthermore, we demonstrated that this resistance mechanism was independent of PGE(2) production as the addition of the specific COX-2 activity inhibitor, NS-398, did not affect apoptosis in UA-treated cells.

A novel form of melanoma apoptosis resistance: Melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.

Sedimentation field-flow fractionation separation of proliferative and differentiated subpopulations during Ca2+-induced differentiation in HaCaT cells

The spontaneously immortalized human keratinocyte cell line HaCaT is widely used as a human keratinocyte model. In a previous comparative study between normal human keratinocytes (NHKs) and HaCaT, we reported that Ca2+ concentrations greater than 1mM induced differentiation in vitro in both cell types, notably characterized by increased expression of differentiation markers keratins 1 (K1), 10 (K10) and involucrin. Surprisingly, cells had a higher proliferative activity than those cultured with low Ca2+ levels. These results raised many questions; in particular concerning the emergence of HaCaT cells subpopulation which would have different differentiation states and/or proliferation rates throughout Ca2+-induced differentiation. To isolate these subpopulations, we used sedimentation field-flow fractionation (SdFFF). Results demonstrated that the most differentiated cells (HC-F1), characterized by the highest expression of keratinocyte differentiation markers, had the lowest proliferative activity. In contrast, less differentiated cells (HC-F2) maintained a higher proliferative activity. SdFFF is a tool to sort differentiated and/or proliferating cells from a total pool previously treated with a Ca2+ concentration inducing differentiation, and can be use to prepare biological models necessary for studying HaCaT cell proliferation after Ca2+-induced differentiation treatment.

Phytochemical potential of Daphne gnidium in inhibiting growth of melanoma cells and enhancing melanogenesis of B16-F0 melanoma

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16‐F0 and B16‐F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti‐proliferative activity after incubation with the two types of tumour skin cells B16‐F0 and B16‐F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2‐M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16‐F0 melanoma cells. Copyright

Resistance to ursolic acid-induced apoptosis through involvement of melanogenesis and COX-2/PGE2 pathways in human M4Beu melanoma cancer cells

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. Previously, we had showed that B16-F0 murine melanoma cells undergoing apoptosis are able to delay their own death induced by ursolic acid (UA), a natural pentacyclic triterpenoid compound. We had demonstrated that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were implicated in an apoptosis resistance mechanism. Several resistance mechanisms to apoptosis have been characterized in melanoma such as hyperactivation of DNA repair mechanisms, drug efflux systems, and reinforcement of survival signals (PI3K/Akt, NF-κB and Raf/MAPK pathways). Otherwise, other mechanisms of apoptosis resistance involving different proteins, such as cyclooxygenase-2 (COX-2), have been described in many cancer types. By using a strategy of specific inhibition of each ways, we suggested that there was an interaction between melanogenesis and COX-2/PGE2 pathway. This was characterized by analyzing the COX-2 expression and activity, the expression of tyrosinase and melanin production. Furthermore, we showed that anti-proliferative and proapoptotic effects of UA were mediated through modulation of multiple signaling pathways including Akt and ERK-1/2 proteins. Our study not only uncovers underlying molecular mechanisms of UA action in human melanoma cancer cells but also suggest its great potential as an adjuvant in treatment and cancer prevention.

Resistance to 3-HTMC-Induced Apoptosis Through Activation of PI3K/Akt, MEK/ERK, and p38/COX-2/PGE2 Pathways in Human HT-29 and HCT116 Colorectal Cancer Cells

Increasing incidence and mortality of colorectal cancer brings the necessity to uncover new possibilities in its prevention and treatment. Chalcones have been identified as interesting compounds having chemopreventive and antitumor properties. In this study, we investigated the effects of the synthetic chalcone derivative 3‐hydroxy‐3′,4,4′,5′‐tetra‐methoxy‐chalcone (3‐HTMC) on proliferation, cell cycle distribution, apoptosis, and its mechanism of action in human colorectal HT‐29 (COX‐2 sufficient) and HCT116 (COX‐2 deficient) cancer cells. We showed that 3‐HTMC decreased cell viability in a dose‐dependent manner with a more potent antiproliferative effect on HCT116 than HT‐29 cells. Flow cytometric analysis revealed G2/M cell cycle accumulation in HT‐29 cells and significant G2/M arrest in HCT116 cells with a subsequent apoptosis shown by appearance of Sub‐G1 peak. We demonstrated that 3‐HTMC treatment on both cell lines induced apoptotic process associated with overexpression of death receptor DR5, activation of caspase‐8 and ‐3, PARP cleavage, and DNA fragmentation. In addition, 3‐HTMC induced activation of PI3K/Akt and MEK/ERK principal survival pathways which delay 3‐HTMC‐induced apoptosis in both cell lines. Furthermore, COX‐2 overexpression in HT‐29 cells contributes to apoptosis resistance which explains the difference of sensitivity between HT‐29 and HCT116 cells to 3‐HTMC treatment. Even if resistance mechanisms to apoptosis reduced chalcone antitumoral potential, our results suggest that 3‐HTMC may be considered as an interesting compound for colorectal cancer therapy or chemoprevention. J. Cell. Biochem. 117: 2875–2885, 2016.

Crataegus azarolus Leaves Induce Antiproliferative Activity, Cell Cycle Arrest, and Apoptosis in Human HT‐29 and HCT‐116 Colorectal Cancer Cells

Limited success has been achieved in extending the survival of patients with metastatic colorectal cancer (CRC). There is a strong need for novel agents in the treatment and prevention of CRC. Therefore, in the present study we evaluated the antiproliferative and pro‐apoptotic potential of Crataegus azarolus ethyl acetate extract in HCT‐116 and HT‐29 human colorectal cancer cell lines. Moreover, we attempted to investigate the signaling pathways that should be involved in its cytotoxic effect. The Crataegus azarolus ethyl acetate extract‐induced growth inhibitory effect was associated with DNA fragmentation, sub‐G1 peak, loss of mitochondrial potential, and poly (ADP‐ribose) polymerase (PARP) cleavage. In addition, ethyl acetate extract of Crataegus azarolus induced the cleavage of caspase‐8. It has no effect on steady‐state levels of total Bcl‐2 protein. Whereas Bax levels decreased significantly in a dose‐dependent manner in both tested cell lines. Taken together, these findings confirm the involvement of the extrinsic pathway of apoptosis. The apoptotic cell death induced by ethyl acetate extract of Crataegus azarolus was accompanied by an enhancement of the p21 expression but not through p53 activation in human colorectal cancer cells. The above‐mentioned data provide insight into the molecular mechanisms of Crataegus azarolus ethyl acetate extract‐induced apoptosis in CRC. Therefore, this compound should be a potential anticancer agent for the treatment of CRC. J. Cell. Biochem. 117: 1262–1272, 2016.