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Gliomas are aggressive brain tumors with very high resistance to chemotherapy throughout the overexpression of multiple intracellular survival pathways. Therefore, the aim of the present study was to investigate for the first time the anticancer activity of LY294002, phosphatidylinositol 3-kinase (PI3K) inhibitor and sorafenib, and rapidly accelerated fibrosarcoma kinase (Raf) inhibitor in the elimination of human glioma cells by programmed cell death. MOGGCCM (anaplastic astrocytoma, III) and T98G (glioblastoma multiforme, IV) cell lines incubated with LY294002 and/or sorafenib were used in the experiments. Simultaneous treatment with both drugs was more effective in the elimination of cancer cells on the way of apoptosis with no significant necrotic effect than single application. It was correlated with decreasing the mitochondrial membrane potential and activation of caspase 3 and 9. The expression of Raf and PI3K was also inhibited. Blocking of those kinases expression by specific siRNA revealed significant apoptosis induction, exceeding the level observed after LY294002 and sorafenib treatment in non-transfected lines but only in MOGGCCM cells. Our results indicated that combination of LY294002 and sorafenib was very efficient in apoptosis induction in glioma cells. Anaplastic astrocytoma cells turned out to be more sensitive for apoptosis induction than glioblastoma multiforme after blocking PI3K and Raf expression with siRNA. Supplementary Information The online version contains supplementary material available at 10.1007/s00441-021-03481-0.

Aim: The aim of the present study was to investigate the efficacy of osthole (7-metoxy-8-isopenthenocoumarin) alone and combined with tamoxifen (TAM) in the elimination of human cervical cancer cells via programmed death. The involvement of heat shock proteins, i.e. well-known molecular chaperones, will be investigated. Material/Methods: Three human cervical cancer cell lines, infected with human papilloma virus (HPV), i.e. HeLa (HPV 18), SiHa (HPV 16), and CaSki (HPV 16 and 18), were used in the experiments. After osthole and TAM treatment, cells stained with fluorochromes were analyzed microscopically according to apoptotic, autophagic, and necrotic morphology. Hsp27, Hsp72, and Hsp90 levels were analyzed by immunoblotting. Transfection with specific siRNA was used for blocking of Hsp expression. Results: In the HeLa, CaSki, and SiHa cell lines, osthole and TAM applied alone had no significant effect on cell death induction. This was correlated with an overexpression of heat shock proteins 27, 72, and 90. In the case of a combination of both drugs, the level of apoptosis was elevated only in SiHa cells. Preincubation with osthole followed by TAM addition as well as simultaneous incubation with both drugs was the most effective. This was correlated with the inhibition of Hsp27, Hsp72, and Hsp90 expression. Blocking of Hsp expression with specific siRNA increased the sensitivity of the studied cell lines to the induction of apoptosis, but not to autophagy or necrosis. Conclusions: Our results indicated that the elimination of heat shock proteins from cervical cancer cells sensitized them to initiation of apoptosis after osthole and tamoxifen treatment.

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.

In the recent years, coumarin bioactive compounds have been identified to posess anticancer properties. Therefore, the aim of the present study was to investigate for the first time the efficacy of osthole, umbelliferone, esculin, and 4-hydroxycoumarin, alone and in combination with Temozolomide, in the elimination of deadly brain tumors, anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) cells via programmed death. Our results indicated that osthole, umbelliferone, esculin, and 4-hydroxycoumarin initiated mainly apoptosis in the T98G and MOGGCCM cells. Osthole was the most effective. It also initiated autophagy in a small percentage of the cell population. The co-incubation with Temozolomide did not increase the pro-apoptotic potential of natural compounds but decreased the level of autophagy in the T98G cells. Apoptosis was associated with reduced mitochondrial membrane potential, activation of caspase 3, inhibition of Bcl-2 expression and the presence of a Bcl-2/Beclin 1. Blocking of Bcl-2 expression resulted in promotion of apoptosis, but not autophagy, in the MOGGCCM and T98G lines. It also sensitized astrocytoma cells, but not GBM, to the combined osthole and TMZ treatment, which was correlated with a reduced level of Beclin 1 and increased expression of caspase 3. Osthole and TMZ, alone and in combination, inhibited the migratory phenotype of the GBM and AA cells. In summary, our results indicated that osthole effectively eliminated glioma cells via apoptosis, what was correlated with Bcl-2/Beclin 1 complex formation. Considering the anti-migratory effect, osthole and Temozolomide display antiglioma potential but it needs further extensive studies.

Simple Summary Flavonoids are plant polyphenolic secondary metabolites, commonly consumed in the human diet. Lensoside Aβ is a quercetin glycoside isolated from the aerial parts of lentil (Lens culinaris) organs. The activity of this secondary metabolite, especially in terms of its anticancer potential, has been poorly studied. Currently, there are no published data about the effect of this flavonoid on gliomas, which are so-far incurable, aggressive neoplasms of the central nervous system with a highly infiltrative character. In this study, we found that lensoside Aβ itself exhibits poor anti-glioma properties but exerts a strongly potentiated effect in combination with sorafenib (inhibitor of Raf kinase) on apoptosis induction in cancer cells. Our results have shown that sorafenib with lensoside Aβ seems to be a promising combination that might be useful in glioma therapy. Additionally, the former observation, pointing to the key role of flavonoids as adjuvants in chemotherapy, is confirmed. Abstract Aim: The anti-glioma effect of lensoside Aβ alone and in combination with sorafenib (pro-survival Raf kinase inhibitor) was evaluated for the first time in terms of programmed cell death induction in anaplastic astrocytoma and glioblastoma multiforme cell lines as an experimental model. Apoptosis, autophagy, and necrosis were identified microscopically (fluorescence and scanning microscopes) and confirmed by flow cytometry (mitochondrial membrane potential MMP and cell death). The expression of apoptotic (caspase 3) and autophagic markers (beclin 1) as well as Raf kinase were estimated by immunoblotting. The FTIR method was used to determine the interaction of the studied drugs with lipid and protein groups within cells, while the modes of drug action within the cells were assessed with the FLIM technique. Results: Lensoside Aβ itself does not exhibit anti-glioma activity but significantly enhances the anti-cancer potential of sorafenib, initiating mainly apoptosis of up to 90% of cells. It was correlated with an increased level of active caspase 3, a reduced MMP value, and a lower level of Raf kinase. The interaction with membrane structures led to morphological changes typical of programmed death. Conclusions: Our results indicate that lensoside Aβ plays an important role as an adjuvant in chemotherapy with sorafenib and may be a potential candidate in anti-glioma combination therapy.

Coumarins, which occur naturally in the plant kingdom, are diverse class of secondary metabolites. With their antiproliferative, chemopreventive and antiangiogenetic properties, they can be used in the treatment of cancer. Their therapeutic potential depends on the type and location of the attachment of substituents to the ring. Therefore, the aim of our study was to investigate the effect of simple coumarins (osthole, umbelliferone, esculin, and 4-hydroxycoumarin) combined with sorafenib (specific inhibitor of Raf (Rapidly Accelerated Fibrosarcoma) kinase) in programmed death induction in human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells lines. Osthole and umbelliferone were isolated from fruits: Mutellina purpurea L. and Heracleum leskowii L., respectively, while esculin and 4-hydroxycoumarin were purchased from Sigma Aldrich (St. Louis, MO, USA). Apoptosis, autophagy and necrosis were identified microscopically after straining with specific fluorochromes. The level of caspase 3, Beclin 1, PI3K (Phosphoinositide 3-kinase), and Raf kinases were estimated by immunoblotting. Transfection with specific siRNA (small interfering RNA) was used to block Bcl-2 (B-cell lymphoma 2), Raf, and PI3K expression. Cell migration was tested with the wound healing assay. The present study has shown that all the coumarins eliminated the MOGGCCM and T98G tumor cells mainly via apoptosis and, to a lesser extent, via autophagy. Osthole, which has an isoprenyl moiety, was shown to be the most effective compound. Sorafenib did not change the proapoptotic activity of this coumarin, however, it reduced the level of autophagy. At the molecular level, the induction of apoptosis was associated with a decrease in the expression of PI3K and Raf kinases, whereas an increase in the level of Beclin 1 was observed in the case of autophagy. Inhibition of the expression of this protein by specific siRNA eliminated autophagy. Moreover, the blocking of the expression of Bcl-2 and PI3K significantly increased the level of apoptosis. Osthole and sorafenib successfully inhibited the migration of the MOGGCCM and T98G cells.

Furanocoumarins are a class of natural compounds produced by several plants, including those consumed by humans. They have been used medicinally in eastern countries for ages. Given the growing body of evidence about their anticancer potential and observations that naturally occurring compounds potentiate the antitumor activity of chemotherapeutics, more attention is paid to elucidation of the nature of furanocoumarins and the possibility of using thereof in practice. The general mechanism by which furanocoumarins eliminate cancer cells is based on cell cycle blockage and initiation of programmed death like apoptosis or autophagy. The precise molecular mechanism of such an action depends on the chemical structure of furanocoumarins, which is based on the furan ring attached to the coumarin backbone in a linear or angular form as well as the type, location, and number of the substituents attached. The review summarizes the current evidence of the antitumor properties of linear and angular furanocoumarins with special emphasis on the molecular mechanism of elimination of cancer cells via apoptosis and autophagy. Negative aspects of the use of coumarins in anticancer therapy will be also discussed especially in the context of their phototoxicity and potential cancerogenic effect.