Some chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features.

Colorectal cancer (CRC) is the second leading cause of death among cancer patients in the Northern countries. CRC can reappear a long time after treatment. Recent clinical studies demonstrated that, in response to chemotherapy, cancer cells may undergo stress-induced premature senescence (SIPS), which typically results in growth arrest. Nonetheless, these senescent cells were reported to divide in an atypical manner and thus contribute to cancer re-growth. Therefore, we examined if SIPS escape may follow treatment with chemotherapeutics used clinically: 5-fluorouracil (5-FU), oxaliplatin (OXA) and irinotecan (IRINO). To mimic the therapeutic regimes we exposed human colon cancer HCT116 and SW480 cells to repeated cycles of drug treatment. The cells treated with 5-FU or IRINO exhibited several hallmarks of SIPS: growth arrest, increased size and granularity, polyploidization, augmented activity of the SA-ß-galactosidase, accumulation of P21 and CYCLIN D1 proteins, and the senescence-associated secretory phenotype. Moreover, re-population of the cancer cell cultures was delayed upon treatment with the senescence-inducing agents. At the same time, we detected a subpopulation of senescent colon cancer cells with features of stemness: elevated NANOG expression, exclusion of Hoechst 33342 (typical for side population) and increased CD24 expression. Additionally, rare, polyploid cells exhibited blastocyst-like morphology and produced progeny. In parallel, majority of chemotherapeutics-treated cells underwent mesenchymal to epithelial transition, as the percentage of CD44-positve cells was reduced, and levels of E-cadherin (epithelial marker) were elevated. Our study demonstrates that a subpopulation of chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features. This may contribute to their resistance to chemotherapy and their ability to re-grow cancer after completion of therapeutic intervention.

Ciprofloxacin inhibits proliferation and promotes generation of aneuploidy in Jurkat cells.

Ciprofloxacin is widely used in antimicrobial therapy. However it also inhibits mitochondrial topoisomerase II and therefore affects cellular energy metabolism. At a concentration exceeding 80 microg/ml ciprofloxacin induces apoptosis, while at 25 microg/ml it inhibits proliferation of Jurkat cells without any symptoms of cell death. The aim of this study was to explain the mechanisms of ciprofloxacin-evoked perturbations of the cell cycle. Human lymphoidal cells (Jurkat) were exposed to ciprofloxacin (25 microg/ml) for 4-11 days and effects of the drug on cell proliferation (light microscopy), cell cycle (flow cytometry), cell size and morphology (confocal microscopy) as well as number of chromosomes (chromosomal spread analysis) were investigated. Exposition of Jurkat cells to ciprofloxacin inhibited cell proliferation,increased proportion of cells in the G2/M-phase of the cell cycle, compromised formation of the mitotic spindle and induced aneuploidy. These observations indicate that ciprofloxacin applied at concentrations insufficient for induction of apoptosis may stop cell proliferation by inhibition of mitosis. Chromosomal instability of such cells may, at least potentially, increase a risk of cancer development.

The promise of slow down ageing may come from curcumin.

No genes exist that have been selected to promote aging. The evolutionary theory of aging tells us that there is a trade-off between body maintenance and investment in reproduction. It is commonly acceptable that the ageing process is driven by the lifelong accumulation of molecular damages mainly due to reactive oxygen species (ROS) produced by mitochondria as well as random errors in DNA replication. Although ageing itself is not a disease, numerous diseases are age-related, such as cancer, Alzheimer's disease, atherosclerosis, metabolic disorders and others, likely caused by low grade inflammation driven by oxygen stress and manifested by increased level of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-alpha, encoded by genes activated by the transcription factor NF-kappaB. It is believed that ageing is plastic and can be slowed down by caloric restriction as well as by some nutraceuticals. As the low grade inflammatory process is believed substantially to contribute to ageing, slowing ageing and postponing the onset of age-related diseases may be achieved by blocking the NF-kappaB-dependent inflammation. In this review we consider the possibility of the natural spice curcumin, a powerful antioxidant, anti-inflammatory agent and efficient inhibitor of NF-kappaB and the mTOR signaling pathway which overlaps that of NF-kappaB, to slow down ageing.

Curcumin induces apoptosis-independent death in oesophageal cancer cells.

BACKGROUND: Oesophageal cancer incidence is increasing and survival rates remain extremely poor. Natural agents with potential for chemoprevention include the phytochemical curcumin (diferuloylmethane). We have examined the effects of curcumin on a panel of oesophageal cancer cell lines. METHODS: MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assays and propidium iodide staining were used to assess viability and DNA content, respectively. Mitotic catastrophe (MC), apoptosis and autophagy were defined by both morphological criteria and markers such as MPM-2, caspase 3 cleavage and monodansylcadaverine (MDC) staining. Cyclin B and poly-ubiquitinated proteins were assessed by western blotting. RESULTS: Curcumin treatment reduces viability of all cell lines within 24 h of treatment in a 5-50 muM range. Cytotoxicity is associated with accumulation in G2/M cell-cycle phases and distinct chromatin morphology, consistent with MC. Caspase-3 activation was detected in two out of four cell lines, but was a minor event. The addition of a caspase inhibitor zVAD had a marginal or no effect on cell viability, indicating predominance of a non-apoptotic form of cell death. In two cell lines, features of both MC and autophagy were apparent. Curcumin-responsive cells were found to accumulate poly-ubiquitinated proteins and cyclin B, consistent with a disturbance of the ubiquitin-proteasome system. This effect on a key cell-cycle checkpoint regulator may be responsible for the mitotic disturbances and consequent cytotoxicity of this drug. CONCLUSION: Curcumin can induce cell death by a mechanism that is not reliant on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of oesophageal cancer.

Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells.

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.

Pantothenic acid protects jurkat cells against ultraviolet light-induced apoptosis.

Human leukemic T lymphocytes (Jurkat cells) were induced to undergo apoptosis by brief irradiation with ultraviolet C light (254 nm). This was accompanied by accumulation of lipid peroxidation products in the form of conjugated dienes, a decrease of total glutathione content, and a shift of its redox state towards the oxidized form. Preincubation of the cells with 1 mM pantothenate resulted in a significant elevation of total glutathione content of the cells, reaching its maximum level, 160% of the control, after 3 h. Similar increase was observed after preincubation with 5 mM N-acetylcysteine, a known precursor of glutathione. Both pantothenic acid and N-acetylcysteine alleviated the ultraviolet-induced decrease of glutathione content, diminished lipid peroxidation, and partly protected the cells against apoptosis produced by ultraviolet irradiation.

Effect of aging on UVC-induced apoptosis of rat splenocytes.

UVC-induced apoptotic symptoms such as morphological changes, DNA fragmentation, Bcl-2 and Bax protein expression were examined in primary splenocyte cultures from young (3 months) and old (24 months) rats. The activities of AP-1 and CRE transcription factors in UVC-irradiated splenocytes were also assessed. At 24 h after UVC irradiation 40% of cells derived from young rats were found to be apoptotic, which was twice as much as in splenocytes from old rats. Apoptosis in cells from old rats did not give typical symptoms like a "DNA ladder" and Bcl-2 protein downregulation, in contrast to splenocytes from young rats. No AP-1 transcription factor activity was found in UVC-irradiated splenocytes from old animals and only a trace activity in splenocytes from young animals. This indicates that, UVC-induced apoptosis in rat splenocytes is practically AP-1 independent and that cells from old rats are less sensitive to UVC irradiation than splenocytes from young rats.

Effect of curcumin on the apoptosis of rodent and human nonproliferating and proliferating lymphoid cells.

Curcumin, a major active component of turmeric, has been recognized as an anticarcinogenic agent because of its propensity to induce apoptosis in vivo and in vitro. Previously, we showed that curcumin protects cells against oligonucleosomal DNA fragmentation and induces a novel apoptosis-like pathway in Jurkat cells (Piwocka et al. Exp Cell Res 249, 299-307, 1999). Here, we have studied the ability of curcumin to induce cell death in other human and rodent transformed as well as normal cells. Normal cells were quiescent or stimulated to proliferate. We showed that 50 microM pigment is able to induce cell death in all studied cells, but cell death symptoms varied for different cells. All the cells died as assessed by the TdT-mediated UTP nick end labeling method or trypan blue exclusion test. No one type of cells showed oligonucleosomal DNA fragmentation (DNA "ladder") due to curcumin action, although in HL-60 cells, we were able to observe sub-G1 formation and caspase-3 activation. Together, these data showed that curcumin induces cell death in all tested cells that can be classified as apoptosis-like, and only in HL-60 cells can it be recognized as classical apoptosis.

UVC-induced cell death of IL-2-dependent human lymphocytes.

We compared the in vitro propensity of human IL-2-dependent lymphocytes (young proliferating and senescent non-proliferating), and resting peripheral blood lymphocytes (PBLs) to undergo UVC-induced apoptosis. The activities of AP-1 (activator protein-1), CRE (cAMP response element) and OCT-1 (octamer-1) transcription factors in all lymphocytes were also assessed. At 24 h after UVC treatment, half of young proliferating T lymphocytes and about a quarter of PBLs and senescent non-proliferating cells were apoptotic, as shown by flow cytometry. However, only in young lymphocytes were both typical DNA 'ladder' and Bcl-2 downregulation evident. The AP-1 transcription factor was activated by UVC in IL-2-dependent young and senescent, but not resting lymphocytes. Taken together, the data show different propensities of resting, proliferating and senescent human lymphocytes to undergo UVC-induced apoptosis and AP-1 activation.

A novel apoptosis-like pathway, independent of mitochondria and caspases, induced by curcumin in human lymphoblastoid T (Jurkat) cells.

We have shown previously [E. Sikora, A. Bielak-Zmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899-907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 microM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Delta psi). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Delta psi, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from "classical" by the lack of mitochondrial depolarization and of the involvement of caspases.

Glutathione-independent mechanism of apoptosis inhibition by curcumin in rat thymocytes.

Curcumin (CUR) is a natural yellow dye with antioxidant and scavenging properties present in Curcuma species. It is widely used as an anti-inflammatory, anti-mutagenic and chemopreventive agent. In addition to its inhibitory effect on proliferation, CUR has recently been shown to block dexamethasone-induced programmed cell death (apoptosis) of rat thymocytes. Because cellular thiols seem to play a role in redox regulation of apoptosis, the mechanism of the anti-apoptotic effect of CUR was studied by examining the levels of glutathione and acid-soluble sulfhydryl groups. CUR was shown to prevent the glutathione loss occurring in dexamethasone-treated thymocytes, enhancing intracellular glutathione content at 8 hr to 192% of that of nontreated cells. A 60% increase in acid-soluble sulfhydryl groups was also observed. In the presence of L-buthionine S,R-sulfoximine (BSO, an inhibitor of glutathione synthesis), intracellular glutathione content of thymocytes treated with dexamethasone and CUR fell to 31% and that of the acid-soluble sulfhydryl groups to 23% of control after 8 hr. Unexpectedly, the electrophoretic and flow cytometric studies of DNA fragmentation demonstrated that apoptosis did not occur even after 20 hr of incubation with buthionine S,R-sulfoximine and dexamethasone, while control thymocytes and the cells treated only with buthionine S,R-sulfoximine showed DNA fragmentation at a level corresponding to spontaneous apoptosis. These results show that CUR treatment elevated the concentrations of glutathione and nonprotein sulfhydryl groups, thus preventing their decrease in apoptotic thymocytes. Coadministration of L-buthionine S,R-sulfoximine and CUR did not affect the anti-apoptotic effect of CUR suggesting a glutathione-independent mechanism of cell protection.

Inhibition of proliferation and apoptosis of human and rat T lymphocytes by curcumin, a curry pigment.

Curcumin (diferuoylmethane), the yellow pigment in the rhizome of tumeric (Curcuma longa), an ingredient of curry spice, is known to exhibit a variety of pharmacological effects including antitumor, antiinflammatory, and antiinfectious activities. Although its precise mode of action remains elusive, curcumin has been shown to suppress the activity of the AP-1 transcription factor in cells stimulated to proliferate. In this study, we observed that curcumin (50 microM) inhibited proliferation of rat thymocytes stimulated with concanavalin A (Con A) as well as that of human Jurkat lymphoblastoid cells in the logarithmic growth phase. The pigment also inhibited apoptosis in dexamethasone-treated rat thymocytes and in UV-irradiated Jurkat cells as judged by DNA ladder formation, cellular morphological changes, and flow cytometry analysis. The inhibition of apoptosis by curcumin in rat thymocytes was accompanied by partial suppression of AP-1 activity. Complete suppression of AP-1 activity was observed in Con A-treated, proliferating thymocytes. The capacity of curcumin to inhibit both cell growth and death strongly implies that these two biological processes share a common pathway at some point and that curcumin affects a common step, presumably involving a modulation of the AP-1 transcription factor.