Carotenoid-Enriched Nanoemulsions and γ-Rays Synergistically Induce Cell Death in a Novel Radioresistant Osteosarcoma Cell Line.

We previously demonstrated that SAOS human osteosarcoma cells, incubated with carotenoid-enriched nanoemulsions (CEN), activated a nonprotective form of autophagy and delayed cell proliferation. The present work focuses on the biological effects of CEN on a derivative of SAOS cells named SAOS400, recently described for their radiation resistance and higher expression of therapy-induced senescence (TIS) markers. SAOS400 cells, incubated with CEN, activated a "cytostatic" form of autophagy confirmed by cell cycle arrest in the G2/M phase and increased expression of autophagic proteins. Treatment of SAOS400 cells with CEN also resulted in decreased expression of the senescence marker p16INK4. However, when SAOS400 cells were γ-irradiated in combination with CEN, the threshold for cell death was reached (>60% after 96 h). We showed that this type of cell death corresponded to 'cytotoxic' or 'lethal' autophagy and that the combined treatment of CEN plus γ-rays was synergistic, with the combination index < 1. Since CEN contained ß-carotene, the pure compound was used in SAOS400 cells at the same concentration present in CEN and up to 10 times higher. However, no radio-sensitizing effect of ß-carotene was observed, suggesting that the biological effect of CEN was due to less abundant but more bioactive molecules, or to the synergistic activity of multiple components present in the extracts, confirming the functional pleiotropy of natural extracts enriched in bioactive molecules.

Biochemical and Cellular Characterization of New Radio-Resistant Cell Lines Reveals a Role of Natural Flavonoids to Bypass Senescence.

Cancer is one of the main causes of death worldwide, and, among the most frequent cancer types, osteosarcoma accounts for 56% of bone neoplasms observed in children and colorectal cancer for 10.2% of tumors diagnosed in the adult population. A common and frequent hurdle in cancer treatment is the emergence of resistance to chemo- and radiotherapy whose biological causes are largely unknown. In the present work, human osteosarcoma (SAOS) and colorectal adenocarcinoma (HT29) cell lines were γ-irradiated at doses mimicking the sub-lethal irradiation in clinical settings to obtain two radio-resistant cellular sub-populations named SAOS400 and HT500, respectively. Since "therapy-induced senescence" (TIS) is often associated with tumor response to radiotherapy in cancer cells, we measured specific cellular and biochemical markers of senescence in SAOS400 and HT500 cells. In detail, both cell lines were characterized by a higher level of expression of cyclin-dependent kinase inhibitors p16INK4 and p21CIP1 and increased positivity to SAß-gal (senescence-associated ß-galactosidase) with respect to parental cells. Moreover, the intracellular levels of reactive oxygen species in the resistant cells were significantly lower compared to the parental counterparts. Subsequently, we demonstrated that senolytic agents were able to sensitize SAOS400 and HT500 to cell death induced by γ-irradiation. Employing two natural flavonoids, fisetin and quercetin, and a BH3-mimetic, ABT-263/navitoclax, we observed that their association with γ-irradiation significantly reduced the expression of p16INK4, p21CIP1 and synergistically (combination index < 1) increased cell death compared to radiation mono-alone treatments. The present results reinforce the potential role of senolytics as adjuvant agents in cancer therapy.

Hypoxia-inducible transgelin 2 selects epithelial-to-mesenchymal transition and γ-radiation-resistant subtypes by focal adhesion kinase-associated insulin-like growth factor 1 receptor activation in non-small-cell lung cancer cells.

Microenvironment, such as hypoxia common to cancer, plays a critical role in the epithelial-to-mesenchymal transition (EMT) program, which is a major route of cancer metastasis and confers γ-radiation resistance to cells. Herein, we showed that transgelin 2 (TAGLN2), an actin-binding protein, is significantly induced in hypoxic lung cancer cells and that Snail1 is simultaneously increased, which induces EMT by downregulating E-cadherin expression. Forced TAGLN2 expression induced severe cell death; however, a small population of cells surviving after forced TAGLN2 overexpression showed γ-radiation resistance, which might promote tumor relapse and recurrence. These surviving cells showed high metastatic activity with an increase of EMT markers including Snail1. In these cells, TAGLN2 activated the insulin-like growth factor 1 receptor ß (IGF1Rß)/PI3K/AKT pathway by recruitment of focal adhesion kinase to the IGF1R signaling complex. Activation of the IGF1Rß/PI3K/AKT pathway also induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which is involved in Snail1 stabilization. Therefore, both the IGF1Rß inhibitor (AG1024) and the PI3K inhibitor (LY294002) or AKT inactivation with MK2206 lower the cellular level of Snail1. Involvement of GSK3ß was also confirmed by treatment with lithium chloride, the inducer of GSK3ß phosphorylation, or MG132, the 26S proteasomal inhibitor, which also stabilized Snail1. In conclusion, the present study provides important evidence that hypoxia-inducible TAGLN2 is involved in the selection of cancer cells with enhanced EMT properties to overcome the detrimental environment of cancer cells.