Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.

Glioblastoma multiforme is the most aggressive brain tumor, and human T98G cells constitute a useful glioblastoma multiforme model to evaluate the chemotherapeutic agents. Modern life (shiftwork, jetlag, etc.) may cause circadian disorganization promoting higher cancer risk and metabolic disorders. Although little is known about the tumor-intrinsic circadian clock function, pharmacological modulation of circadian components may offer selective anticancer strategies. REV-ERBs are heme-binding circadian clock components acting as repressors of processes involved in tumorigenesis such as metabolism, proliferation, and inflammation. A synthetic pyrrole derivative (SR9009) that acts as REV-ERBs-specific agonists exhibits potent in vivo activity on metabolism and tumor cell viability. Here, we investigated SR9009 effects on T98G cell viability, differential chemotherapy time responses, and underlying metabolic processes (reactive oxygen species [ROS] and lipid droplets [LDs]) and compared it with the proteasome inhibitor Bortezomib treatment. SR9009-treated cells exhibited significant reduction in cell viability with consequences on cell cycle progression. Dexamethasone synchronized cells displayed differential time responses to SR9009 treatment with highest responses 18 to 30 h after synchronization. SR9009 treatment decreased ROS levels while Bortezomib increased them. However, both treatments significantly increased LD levels, whereas the combined treatment showed additive or synergistic effects between both drugs. In addition, we extended these studies to HepG2 cells which also showed a significant decrease in cell viability and ROS levels and the increase in LD levels after SR9009 treatment. Our results suggest that the pharmacological modulation of the tumor-intrinsic clock by REV-ERB agonists severely affects cell metabolism and promotes cytotoxic effects on cancer cells.

Tumors of the nervous system including glioblastoma multiforme (GBM) are the most frequent and aggressive form of brain tumors; however, little is known about the impact of the circadian timing system on the formation, growth, and treatment of these tumors. We investigated day/night differences in tumor growth after injection of A530 glioma cells isolated from malignant peripheral nerve sheath tumor (MPNSTs) of NPcis (Trp53 +/− ; Nf1 +/− ) mice. Synchronized A530 cell cultures expressing typical glial markers were injected at the beginning of the day or night into the sciatic nerve zone of C57BL/6 mice subject to a 12:12 hours light/dark (LD) cycle or after being released to constant darkness (DD). Tumors generated in animals injected early at night in the LD cycle or in DD showed higher growth rates than in animals injected diurnally. No differences were found when animals were injected at the same time with cultures synchronized 12 hours apart. Similar experiments performed with B16 melanoma cells showed higher tumor growth rates in animals injected at the beginning of the night compared to those injected in the daytime. A higher tumor growth rate than that in controls was observed when mice were injected with knocked-down clock gene Bmal1 cells. Finally, when we compared day/ night administration of different doses of the proteasome inhibitor Bortezomib (0.5-1.5 mg/kg) in tumor-bearing animals, we found that low-dose chemotherapy displayed higher efficacy when administered at night. Results suggest the existence of a precise temporal control of tumor growth and of drug efficacy in which the host state and susceptibility are critical. K E Y W O R D SBortezomib, cancer chronotherapy, circadian clock, light/dark differences, tumor growth 2 of 16 | WAGNER Et Al.

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Gliomas are solid tumors of the central nervous system (CNS) that originated from different glial cells. The World Health Organization (WHO) classifies these tumors into four groups (I–IV) with increasing malignancy. Glioblastoma (GBM) is the most common and aggressive type of brain tumor classified as grade IV. GBMs are resistant to conventional therapies with poor prognosis after diagnosis even when the Stupp protocol that combines surgery and radiochemotherapy is applied. Nowadays, few novel therapeutic strategies have been used to improve GBM treatment, looking for higher efficiency and lower side effects, but with relatively modest results. The circadian timing system temporally organizes the physiology and behavior of most organisms and daily regulates several cellular processes in organs, tissues, and even in individual cells, including tumor cells. The potentiality of the function of the circadian clock on cancer cells modulation as a new target for novel treatments with a chronobiological basis offers a different challenge that needs to be considered in further detail. The present review will discuss state of the art regarding GBM biology, the role of the circadian clock in tumor progression, and new chrono-chemotherapeutic strategies applied for GBM treatment.

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4Glioblastoma multiforme is the most aggressive brain tumor; however, little is known about the impact of the 5 circadian system on the tumor formation, growth and treatment. We investigated day/night differences in tumor 6 growth after injecting A530 glioma cells isolated from malignant peripheral nerve sheath tumor of NPcis 7 (Trp53 +/-; Nf1 +/-) mice. Tumors generated in the sciatic nerve zone of C57BL/6 mice injected early at night in 8 the light/dark cycle or in constant darkness, showed higher growth rates than in animals injected diurnally. 9Similar nocturnal increases were observed when injecting B16 melanoma cells or when 10 mice received knocked-down clock gene Bmal1 cells. Moreover, treatment with a low-dose of the proteasome 11 inhibitor Bortezomib (0.5 mg/kg) in tumor-bearing animals, displayed higher efficacy when administered at 12 night. Results suggest the existence of a precise temporal control of tumor growth and of drug efficacy in which 13 the host state and susceptibility are critical. 14 15At the molecular level, the circadian clock operates through a transcriptional/translational feedback loop that 1 involves a group of regulatory transcription factors -clock genes and proteins-including Clock, Bmal1, NPAS2, 2 Periods (Per) and Cryptochromes (Cry), among others [5,6]. Once translated, these proteins display activity as 3 activators (CLOCK, BMAL1, NPAS2) or repressors (PER, CRY) of gene transcription and are capable of 4 driving clock gene rhythms and rhythmic clock-controlled gene expression under a 24 h cycle through a 5 consensus sequence E-box at the promoters of target genes [5]. A secondary cycle involves the nuclear receptor 6 REV-ERBα/β (REV): when expression levels of this receptor are high, it binds to RORE sites present in 7 promoter and enhancer regions of the Clock and Bmal1 genes, repressing its transcription. In contrast, when the 8 REV-ERBs levels are low, another nuclear receptor of retinoic acid, ROR, binds to the RORE site of promoters 9 and may activate Clock and Bmal1 transcription [7,8]. Per1 and 2 are involved in the synchronization of the 10 molecular clock after stimulation by early response transcription factors whose activity is controlled by 11 extracellular signals such as hormones, second messengers, temperature or neurotransmitters. Among these 12 transcription factors are cAMP response elements and protein-CRE binding (CREB) elements, HSF1 that binds 13 to heat shock response elements (HSEs), serum response factors (SRF) that bind to response elements serum 14 (SREs) and glucocorticoid receptors (GR) that bind to glucocorticoid response elements (GREs) [9]. At the 15 cellular level, a redox / metabolic clock has been reported to drive peroxiredoxin (PRX) oxidation cycles and 16shown to be highly conserved through evolution. These redox oscillators are present in all kingdoms of life and 17 even when they interact with the transcriptional molecular clock they can still function in the absence of 18 transcription, as occurs in enucleated cells [10]. 19Through t...

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