Long noncoding RNA growth arrest-specific 5 facilitates glioma cell sensitivity to cisplatin by suppressing excessive autophagy in an mTOR-dependent manner

Malignant glioma is a severe type of brain tumor with a grim prognosis. The occurrence of resistance compromises the efficacy of chemotherapy for glioma. Long noncoding RNA growth arrest-specific 5 (GAS5) has recently become an attractive target for cancer therapy by regulating cell growth, invasion, and migration. Nevertheless, its role in glioma chemoresistance remains elusive. In the current study, the expression of GAS5 was decreased in glioma cell lines, and lower levels of GAS5 were observed in U138 and LN18 glioma cells that had low sensitivity to cisplatin. Functional assay confirmed that knockdown of GAS5 enhanced cell resistance to cisplatin in U87 cells, which had a relatively high expression of GAS5. Conversely, elevation of GAS5 increased cell sensitivity to cisplatin in U138 cells that had a relatively low expression of GAS5. Mechanistically, cisplatin exposure evoked excessive autophagy concomitant with an increase in autophagy-related LC3II expression and a decrease in autophagy substrate p62 expression, which was reversely muted after GAS5 overexpression. In addition, GAS5 restored cisplatin-inhibited mammalian target of rapamycin (mTOR) activation. Preconditioning with mTOR antagonist rapamycin engendered not only mTOR inhibition but also abrogated GAS5-mediated depression in cisplatin-evoked autophagy. Notably, blocking the mTOR pathway also attenuated GAS5-increased sensitivity to cisplatin in U138 cells. Cumulatively, these findings indicate that GAS5 may blunt the resistance of glioma cells to cisplatin by suppressing excessive autophagy through the activation of mTOR signaling, implying a promising therapeutic strategy against chemoresistance in glioma.     

The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation

Although DNA damaging agents have revolutionized chemotherapy against solid tumors, a narrow therapeutic window combined with severe side effects has limited their broader use. Here we show that RAD001 (everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53, but not mutant p53 tumor cells. The use of isogenic tumor cell lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. We further show that RAD001 sensitizes cells to cisplatin by inhibiting p53-induced p21 expression. Unexpectedly, this effect is attributed to a small but significant inhibition of p21 translation combined with its short half-life. These findings provide the molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors.     

Weak p53 permits senescence during cell cycle arrest

Cell cycle arrest coupled with hyper-active mTOR leads to cellular senescence. While arresting cell cycle, high levels of p53 can inhibit mTOR (in some cell lines), thus causing reversible quiescence instead of senescence. Nutlin-3a-induced p53 inhibited mTOR and thus caused quiescence in WI-38 cells. In contrast, while arresting cell cycle, the DNA-damaging drug doxorubicin (DOX) did not inhibit mTOR and caused senescence. Super-induction of p53 by either nutlin-3a or high concentrations of DOX (high-DOX) prevented low-DOX-induced senescence, converting it into quiescence. This explains why in order to cause senescence, DNA damaging drugs must be used at low concentrations, which arrest cell cycle but do not induce p53 at levels sufficient to suppress mTOR. Noteworthy, very prolonged treatment with nutlin-3a also caused senescence preventable by rapamycin. In RPE cells, low concentrations of nutlin-3a caused a semi-senescent morphology. Higher concentrations of nutlin-3a inhibited mTOR and caused quiescent morphology. We conclude that low p53 levels during prolonged cell cycle arrest tend to cause senescence, whereas high levels of p53 tend to cause either quiescence or cell death.     

In Vitro and In Vivo Antitumor Activity of [Pt(O,O′-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O′-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKC-ε activation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC–siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.     

p53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3′-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.     

Sustained activation of the JNK cascade and rapamycin-induced apoptosis are suppressed by p53/p21(Cip1)

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21(Cip1)arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21(Cip1) independent of cell cycle arrest. Rapamycin induces p21(Cip1) binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21(Cip1).     

mTOR activation induces tumor suppressors that inhibit leukemogenesis and deplete hematopoietic stem cells after Pten deletion

Pten deficiency depletes hematopoietic stem cells (HSCs) but expands leukemia-initiating cells, and the mTOR inhibitor, rapamycin, blocks these effects. Understanding the opposite effects of mTOR activation on HSCs versus leukemia-initiating cells could improve antileukemia therapies. We found that the depletion of Pten-deficient HSCs was not caused by oxidative stress and could not be blocked by N-acetyl-cysteine. Instead, Pten deletion induced, and rapamycin attenuated, the expression of p16(Ink4a) and p53 in HSCs, and p19(Arf) and p53 in other hematopoietic cells. p53 suppressed leukemogenesis and promoted HSC depletion after Pten deletion. p16(Ink4a) also promoted HSC depletion but had a limited role suppressing leukemogenesis. p19(Arf) strongly suppressed leukemogenesis but did not deplete HSCs. Secondary mutations attenuated this tumor suppressor response in some leukemias that arose after Pten deletion. mTOR activation therefore depletes HSCs by a tumor suppressor response that is attenuated by secondary mutations in leukemogenic clones.     

Light‐emitting diode irradiation induces AKT/mTOR‐mediated apoptosis in human pancreatic cancer cells and xenograft mouse model

The beneficial effects of light‐emitting diode (LED) irradiation have been reported in various pathologies, including cancer. However, its effect in pancreatic cancer cells remains unclear. Herein, we demonstrated that blue LED of 460 nm regulated pancreatic cancer cell proliferation and apoptosis by suppressing the expression of apoptosis‐related factors, such as mutant p53 and B‐cell lymphoma 2 (Bcl‐2), and decreasing the expression of RAC‐β serine/threonine kinase 2 (AKT2), the phosphorylation of protein kinase B (AKT), and mammalian target of rapamycin (mTOR). Blue LED irradiation also increased the levels of cleaved poly‐(ADP‐ribose) polymerase (PARP) and caspase‐3 in pancreatic cancer cells, while it suppressed AKT2 expression and inhibited tumor growth in xenograft tumor tissues. In conclusion, blue LED irradiation suppressed pancreatic cancer cell and tumor growth by regulating AKT/mTOR signaling. Our findings indicated that blue LEDs could be used as a nonpharmacological treatment for pancreatic cancer.     

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B" regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.     

Constitutive mTOR activation in TSC mutants sensitizes cells to energy starvation and genomic damage via p53

Miscoordination of growth and proliferation with the cellular stress response can lead to tumorigenesis. Mammalian target of rapamycin (mTOR), a central cell growth controller, is highly activated in some malignant neoplasms, and its clinical implications are under extensive investigation. We show that constitutive mTOR activity amplifies p53 activation, in vitro and in vivo, by stimulating p53 translation. Thus, loss of TSC1 or TSC2, the negative regulators of mTOR, results in dramatic accumulation of p53 and apoptosis in response to stress conditions. In other words, the inactivation of mTOR prevents cell death by nutrient stress and genomic damage via p53. Consistently, we also show that p53 is elevated in TSC tumors, which rarely become malignant. The coordinated relationship between mTOR and p53 during cellular stress provides a possible explanation for the benign nature of hamartoma syndromes, including TSC. Clinically, this also suggests that the efficacy of mTOR inhibitors in anti-neoplastic therapy may also depend on p53 status, and mTOR inhibitors may antagonize the effects of genotoxic chemotherapeutics.     

Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

Twist1 is highly expressed in primary and metastatic non-small cell lung cancer (NSCLC), and thus acts as a critical target for lung cancer chemotherapy. In the current study, we investigated the underlying mechanism initiated by silencing of Twist1 that sensitizes NSCLC cells to cisplatin. Silencing of Twist1 triggered ATP depletion, leading to AMP-activated protein kinase (AMPK)-activated mammalian target of rapamycin (mTOR) inhibition in NSCLC cells. AMPK-induced mTOR inhibition, in turn, resulted in downregulation of ribosome protein S6 kinase 1 (S6K1) activity. Downregulation of mTOR/S6K1 reduced Mcl-1 protein expression, consequently promoting sensitization to cisplatin. Overexpression of Mcl-1 reduced PARP cleavage induced by cisplatin and Twist1 siRNA, suggesting that this sensitization is controlled through Mcl-1 expression. Interestingly, cells treated with Twist1 siRNA displayed upregulation of p21^Waf1/CIP1, and suppression of p21^Waf1/CIP1 with specific siRNA further enhanced the cell death response to cisplatin/Twist1 siRNA. In conclusion, silencing of Twist1 sensitizes lung cancer cells to cisplatin via stimulating AMPK-induced mTOR inhibition, leading to a reduction in Mcl-1 protein. To our knowledge, this is the first report to provide a rationale for the implication of cross-linking between Twist1 and mTOR signaling in resistance of NSCLC to anticancer drugs.     

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70(S6K) in HepG2 and HepG2 cells overexpressing constitutively active Akt/PKB

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70(S6K)). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70(S6K) (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70(S6K) in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70(S6K) in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r(2)=0.525, p<0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.     

Dihydroartemisinin potentiates the anticancer effect of cisplatin via mTOR inhibition in cisplatin-resistant ovarian cancer cells: involvement of apoptosis and autophagy

Dihydroartemisinin (DHA) exhibits anticancer activity in tumor cells but its mechanism of action is unclear. Cisplatin (DDP) is currently the best known chemotherapeutic available for ovarian cancer. However, tumors return de novo with acquired resistance over time. Mammalian target of rapamycin (mTOR) is an important kinase that regulates cell apoptosis and autophagy, and its dysregulation has been observed in chemoresistant human cancers. Here, we show that compared with control ovarian cancer cells (SKOV3), mTOR phosphorylation was abnormally activated in cisplatin-resistant ovarian cancer cells (SKOV3/DDP) following cisplatin monotherapy. Treatment with cisplatin combined with DHA could enhance cisplatin-induced proliferation inhibition in SKOV3/DDP cells. This mechanism is at least partially due to DHA deactivation of mTOR kinase and promotion of apoptosis. Although autophagy was also induced by DHA, the reduced cell death was not found by suppressing autophagic flux by Bafilomycin A1 (BAF). Taken together, we conclude that inhibition of cisplatin-induced mTOR activation is one of the main mechanisms by which DHA dramatically promotes its anticancer effect in cisplatin-resistant ovarian cancer cells.     

Elimination of proliferating cells unmasks the shift from senescence to quiescence caused by rapamycin

Background

Depending on cellular context, p53-inducing agents (such as nutlin-3a) cause different outcomes including reversible quiescence and irreversible senescence. Inhibition of mTOR shifts the balance from senescence to quiescence. In cell lines with incomplete responses to p53, this shift may be difficult to document because of a high proportion of proliferating cells contaminating arrested (quiescent and senescent) cells. This problem also complicates the study of senescence caused by minimal levels of p21 that are capable to arrest a few cells.

Methodology

During induction of senescence by low levels of endogenous p53 and ectopic p21, cells were co-treated with nocodazole, which eliminated proliferating cells. As a result, only senescent and quiescent cells remained.

Results and Discussion

This approach revealed that rapamycin efficiently converted nutlin-induced-senescence into quiescence. In the presence of rapamycin, nutlin-arrested MCF-7 cells retained the proliferative potential and small/lean morphology. Using this approach, we also unmasked senescence in cells arrested by low levels of ectopic p21, capable to arrest only a small proportion of HT1080-p21-9 cells. When p21 did cause arrest, mTOR caused senescent phenotype. Rapamycin and high concentrations of nutlin-3a, which inhibit the mTOR pathway in these particular cells, suppressed senescence, ensuring quiescence instead. Thus, p21 causes senescence passively, just by causing arrest, while still active mTOR drives senescent phenotype.     

Deoxyribose protects against rapamycin-induced cytotoxicity in colorectal cancer cells in vitro

Thymidine phosphorylase (TPase) is also known as the platelet-derived endothelial cell growth factor (PD-ECGF) and plays a role in angiogenesis. Deoxyribose (dR; a downstream TPase-product) addition to endothelial cells may stimulate FAK and p70/S6k signaling, which can be inhibited by rapamycin. Rapamycin is a specific mammalian target of the rapamycin (mTOR) inhibitor, a kinase that lies directly upstream of p70/S6k. This suggests a role for TPase in the mTOR/p70/S6k pathway. In order to study this in more detail, we exposed cells with and without TPase expression to dR and rapamycin and determined the effect on cell growth. We observed protection in cytotoxicity in Colo320 cells, but not Colo320 TP1 cells. This was in part mediated by activation of p70/S6k and inhibition of autophagy. Further studies are recommended to elucidate the mechanism behind the protective effect of dR.     

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.     

Antiproliferative effect of ferrocifen drug candidates on malignant pleural mesothelioma cell lines

The purpose of this study was to investigate the antiproliferative potential of two novel bio-organometallic drug candidates, based on hydroxyl-phenyl-but-1-ene skeleton and containing the ferrocenyl (Fc) moiety, namely ferrociphenol (Fc-diOH) and ferrocifen (Fc-OH-TAM), on two cell lines, named BR95 (epithelial-like) and MM98 (sarcomatous-like), obtained from pleural effusions of previously untreated malignant pleural mesothelioma (MPM) patients. In vitro chemosensitivity of MPM cells towards the title compounds was evaluated by cell viability assay, alkaline Single Cell Gel Electrophoresis (Comet test) and western blotting evaluation of p53 induction. The two bio-organometallic derivatives were found to be more potent in inhibiting cell proliferation than the reference metallo-drug cisplatin (CDDP). This antiproliferative effect cannot be attributed to estrogenic/antiestrogenic activity, since both cell lines resulted to be estrogen insensitive (ER−). Fc-diOH and CDDP were able to upregulate wild type p53 present in MM98 cell line, while Fc-OH-TAM was not. Similarly, Fc-diOH and CDDP induced early DNA damage, while Fc-OH-TAM did not. This indicates that, albeit the similar structures, the two ferrocifens exhert different mechanisms of cytotoxicity on MPM cells.     

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70 in HepG2 and HepG2 cells overexpressing constitutively active Akt/PKB

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70^S6K). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70^S6K (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70^S6K in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70^S6K in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r² = 0.525, p < 0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.