Are Gait Patterns during In-Lab Running Representative of Gait Patterns during Real-World Training? An Experimental Study.

Biomechanical assessments of running typically take place inside motion capture laboratories. However, it is unclear whether data from these in-lab gait assessments are representative of gait during real-world running. This study sought to test how well real-world gait patterns are represented by in-lab gait data in two cohorts of runners equipped with consumer-grade wearable sensors measuring speed, step length, vertical oscillation, stance time, and leg stiffness. Cohort 1 (N = 49) completed an in-lab treadmill run plus five real-world runs of self-selected distances on self-selected courses. Cohort 2 (N = 19) completed a 2.4 km outdoor run on a known course plus five real-world runs of self-selected distances on self-selected courses. The degree to which in-lab gait reflected real-world gait was quantified using univariate overlap and multivariate depth overlap statistics, both for all real-world running and for real-world running on flat, straight segments only. When comparing in-lab and real-world data from the same subject, univariate overlap ranged from 65.7% (leg stiffness) to 95.2% (speed). When considering all gait metrics together, only 32.5% of real-world data were well-represented by in-lab data from the same subject. Pooling in-lab gait data across multiple subjects led to greater distributional overlap between in-lab and real-world data (depth overlap 89.3-90.3%) due to the broader variability in gait seen across (as opposed to within) subjects. Stratifying real-world running to only include flat, straight segments did not meaningfully increase the overlap between in-lab and real-world running (changes of <1%). Individual gait patterns during real-world running, as characterized by consumer-grade wearable sensors, are not well-represented by the same runner's in-lab data. Researchers and clinicians should consider "borrowing" information from a pool of many runners to predict individual gait behavior when using biomechanical data to make clinical or sports performance decisions.

Leg Stiffness, Joint Stiffness, and Running-Related Injury: Evidence From a Prospective Cohort Study.

BACKGROUND: The spring-like behavior of the leg and the joints of the lower body during running are thought to influence a wide range of physiologic and mechanical phenomena, including susceptibility to overuse injury. If leg and joint stiffness are associated with running-related injuries, altering joint or leg stiffness may be a useful avenue for injury rehabilitation and injury prevention programs. PURPOSE: To test the associations between running-related injury and leg stiffness, knee stiffness, and ankle stiffness in a prospective study of recreational runners. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 49 healthy recreational runners took part in a year-long study. Participants completed a 3-dimensional kinematic and kinetic biomechanical assessment at baseline and reported training volume and injury status in a weekly survey during the follow-up period. Relationships between stiffness and injury were assessed at the level of individual legs (n = 98) using spline terms in Cox proportional hazards models. RESULTS: During follow-up, 23 participants (29 legs) sustained injury. The median time to injury was 27 weeks (53.27 hours of training). Relative injury rate as a function of knee stiffness displayed a weak and nonsignificant U-shaped curve (P = .187-.661); ankle and leg stiffness displayed no discernable associations with relative injury rate (leg stiffness, P = .215-.605; ankle stiffness, P = .419-.712). CONCLUSION: Leg and joint stiffness may not be important factors in the development of running-related injuries. Moderate changes in leg and joint stiffness are unlikely to substantially alter injury risk.

CARL: a running recognition algorithm for free-living accelerometer data.

Wearable accelerometers hold great promise for physical activity epidemiology and sports biomechanics. However, identifying and extracting data from specific physical activities, such as running, remains challenging.Objective. To develop and validate an algorithm to identify bouts of running in raw, free-living accelerometer data from devices worn at the wrist or torso (waist, hip, chest).Approach. The CARL (continuous amplitude running logistic) classifier identifies acceleration data with amplitude and frequency characteristics consistent with running. The CARL classifier was trained on data from 31 adults wearing accelerometers on the waist and wrist, then validated on free-living data from 30 new, unseen subjects plus 166 subjects from previously-published datasets using different devices, wear locations, and sample frequencies.Main results. On free-living data, the CARL classifier achieved mean accuracy (F1score) of 0.984 (95% confidence interval 0.962-0.996) for data from the waist and 0.994 (95% CI 0.991-0.996) for data from the wrist. In previously-published datasets, the CARL classifier identified running with mean accuracy (F1score) of 0.861 (95% CI 0.836-0.884) for data from the chest, 0.911 (95% CI 0.884-0.937) for data from the hip, 0.916 (95% CI 0.877-0.948) for data from the waist, and 0.870 (95% CI 0.834-0.903) for data from the wrist. Misclassification primarily occurred during activities with similar torso acceleration profiles to running, such as rope jumping and elliptical machine use.Significance. The CARL classifier can accurately identify bouts of running as short as three seconds in free-living accelerometry data. An open-source implementation of the CARL classifier is available atgithub.com/johnjdavisiv/carl.

Monitoring Gait Complexity as an Indicator for Running-Related Injury Risk in Collegiate Cross-Country Runners: A Proof-of-Concept Study.

Dynamical systems theory suggests that studying the complexity of biological signals could lead to a single gait metric that reliably predicts risk of running-related injury (RRI). The purposes of this pilot study were to examine center of mass (COM) acceleration complexity at baseline, prior to RRI, and the change between timepoints between collegiate runners who developed RRI during a competitive season and those who remained uninjured, and to determine if complexity at these timepoints was associated with increased odds of RRI. Twenty-two collegiate runners from the same cross-country team wore a waist-mounted triaxial accelerometer (100 Hz) during easy-intensity runs throughout the competitive season. RRIs requiring medical attention were reported via an online survey. Control entropy was used to estimate the complexity of the resultant COM acceleration recorded during each run. Associations between complexity and RRI were assessed using a frequency-matching strategy where uninjured participants were paired with injured participants using complexity from the most time-proximal run prior to RRI. Seven runners sustained an RRI. No significant differences were observed between injured and uninjured groups for baseline complexity (p = 0.364, d = 0.405), pre-injury complexity (p = 0.258, d = 0.581), or change from baseline to pre-injury (p = 0.101, d = 0.963). There were no statistically significant associations found between complexity and RRI risk. Although no significant associations were found, the median effect from the models indicated that an increase in baseline complexity, pre-injury complexity, and change in complexity from baseline each corresponded to an increased odds of sustaining an RRI [baseline: odds ratio (OR) = 1.560, 95% CI = 0.587-4.143, p = 0.372; pre-injury: OR = 1.926, 95% CI: 0.689-5.382, p = 0.211; change from baseline: OR = 1.119; 95% CI: 0.839-1.491, p = 0.445). Despite non-significance and wide confidence intervals that included both positive and negative associations, the point estimates for >98% of the 10,000 frequency-case-control-matched model fits indicated that matching strategy did not influence the directionality of the association estimates between complexity and RRI risk (i.e., odds ratio >1.0). This pilot study demonstrates initial feasibility that additional research may support COM acceleration complexity as a useful single-metric monitoring system for RRI risk during real-world training. Follow-up work should assess longitudinal associations between gait complexity and running-related injury in larger cohorts.

Injured Runners Do Not Replace Lost Running Time with Other Physical Activity.

Running-related injuries are common and may pose a barrier to maintaining high levels of overall physical activity. PURPOSE: The aim of this study was to determine whether recreational runners remain physically active while experiencing running-related pain or running-related injury. METHODS: Recreational runners (n = 49) participated in a year-long observational cohort study. Subjects were issued a commercial activity monitor to measure daily physical activity level, quantified by the total minutes of moderate to vigorous physical activity (MVPA). Subjects also completed a weekly survey inquiring about running-related pain and any modifications made to planned running sessions. A week was classified as an "injured week" if a runner reported a reduction or cancellation of at least three planned training sessions, otherwise, it was categorized as an "uninjured week." Separately, pain level was assessed for each week using a 0 to 10 scale. Survey responses were used to longitudinally track pain levels and injury status for each runner. Mixed-effect linear models were used to quantify whether sustaining an injury or reporting running-related pain during a given week were associated with changes in MVPA levels for that week. RESULTS: Compared with uninjured weeks, runners engaged in 14.1 fewer minutes of MVPA per day (95% confidence interval, -22.5 to -6.0) during weeks in which they reported a running-related injury. Lost MVPA during injured weeks was primarily replaced by sedentary activity. There was no significant association between running-related pain and MVPA. CONCLUSIONS: Injured runners do not replace lost running time with other forms of MVPA. Running-related injury and running-related pain should not be conflated; although reporting injury is associated with a reduction in physical activity in recreational runners, high pain levels are not.

Activation of sterile20-like kinase 1 in proteasome inhibitor bortezomib-induced apoptosis in oncogenic K-ras-transformed cells.

Bortezomib (PS-341), a specific proteasome inhibitor, exhibits antitumor activity against a wide range of malignancies. However, the molecular mechanisms by which bortezomib causes apoptosis selectively in cancer cells still remain unclear. Ras signaling is involved in multiple cellular processes, including cell cycle progression, differentiation, and apoptosis, and can either promote or inhibit apoptosis depending on the type of apoptotic stimuli and the cell model. Here, we investigated the role of K-ras signaling in bortezomib-induced apoptosis. We found that K-ras-transformed cells were more susceptible to bortezomib-induced apoptosis than were nontransformed cells and that bortezomib-induced apoptosis was mainly caspase dependent in K-ras-transformed cells. We also found that mammalian sterile20-like kinase 1 (MST1) was activated by bortezomib in K-ras-transformed cells and K-ras-mutated cancer cells. Treatment of K-ras-transformed cells with bortezomib resulted in translocation of MST1 from cytoplasm into the nucleus and an increase of phosphorylated histone H2B and histone H2AX. Moreover, pretreatment with leptomycin B, an inhibitor of the nuclear export signal receptor, dramatically enhanced bortezomib-mediated MST1 activation, phosphorylation of histones H2B and H2AX, and apoptosis induction in K-ras-transformed cells. Knockdown of MST1 expression by small interfering RNA diminished bortezomib-induced apoptosis or caspase-3 activation. Our data suggested that bortezomib may be useful for treatment of K-ras-mutated cancer cells, and MST1 is one of the mediators for bortezomib-induced apoptosis in K-ras-transformed cells.

Eliminating established tumor in nu/nu nude mice by a tumor necrosis factor-alpha-related apoptosis-inducing ligand-armed oncolytic adenovirus.

PURPOSE: The tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector. EXPERIMENTAL DESIGN: We constructed a TRAIL-expressing oncolytic adenovector designated as Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity, and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors. RESULTS: Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing, replication-defective adenovector or an oncolytic adenovector-expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all s.c. xenograft tumors established from a human non-small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term, tumor-free survival. Furthermore, we found no treatment-related toxicity. CONCLUSIONS: Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy.

Identification of a novel synthetic thiazolidin compound capable of inducing c-Jun NH2-terminal kinase-dependent apoptosis in human colon cancer cells.

Development of new therapeutic agents for colon cancer is highly desirable. To this end, we screened a chemical library for new anticancer agents and identified a synthetic compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin (DBPT), which kills cancer cells more effectively than it kills normal human fibroblasts. The molecular mechanism of the antitumor action of DBPT was further analyzed in three human colorectal cancer cell lines. DBPT effectively inhibited the growth of colorectal cancer cells, independent of p53 and P-glycoprotein status, whereas normal fibroblasts were unaffected at the same IC50. Over time, DLD-1 cancer cells treated with DBPT underwent apoptosis. The general caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone partially blocked DBPT-induced apoptosis in a dose-dependent manner. DBPT-induced apoptosis, including cytochrome c release and caspase activation, was abrogated when c-Jun NH2-terminal kinase (JNK) activation was blocked with either a specific JNK inhibitor or a dominant-negative JNK1 gene. However, constitutive JNK activation alone did not replicate the effects of DBPT in DLD-1 cells, and excessive JNK activation by adenovirus encoding MKK7 had little influence on DBPT-induced apoptosis. Our results suggested that DBPT induces apoptosis in colorectal cancer cell lines through caspase-dependent and caspase-independent pathways and that JNK activation was crucial for DBPT-induced apoptosis. DBPT and its analogues might be useful as anticancer agents.

Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS-341, Velcade) and other proteasome inhibitors.

Proteasome inhibitors have emerged as promising anticancer therapeutic agents. Bortezomib (PS-341), a specific proteasome inhibitor, exhibits antitumor activity against a wide range of malignancies and has been approved by the US Food and Drug Administration for the treatment of relapsed or refractory multiple myeloma. However, the molecular mechanisms of bortezomib-mediated apoptosis remain unclear. To characterize the mechanisms of apoptosis induction by proteasome inhibitors, we examined levels of Bcl-2 protein family members (Bik/NBK, Bax, Bak, Bcl-2, and Bcl-XL), release of cytochrome c, and activation of caspase-9 and -3 in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human lung cancer cell line H1299; and human ovarian cancer cell line SKOV3 after they were treated with bortezomib. The result showed that bortezomib induced rapid accumulation of Bik/NBK but not other Bcl-2 family members in all six cell lines. Bortezomib-mediated Bik/NBK accumulation and apoptosis were also observed in human embryonic kidney cells 293 and normal human bronchial epithelial cells. Moreover, dramatic Bik/NBK accumulation and apoptosis induction were observed when cells were treated with proteasome inhibitor MG132 and calpain inhibitor I (ALLN). Furthermore, no detectable changes in IkappaBalpha levels or in NFkappaB functionality were found after treatment with bortezomib. Finally, Bik/NBK accumulation was caused by stabilization of the protein from degradation and was associated with bortezomib cytotoxicity and apoptosis induction. Pretreatment of DLD1 cells with Bik/NBK siRNA reduced bortezomib-mediated Bik/NBK accumulation and cell death. Our results suggested that Bik/NBK is one of the mediators of proteasome inhibitor-induced apoptosis.

Downregulation of XIAP and induction of apoptosis by the synthetic cyclin-dependent kinase inhibitor GW8510 in non-small cell lung cancer cells.

Small-molecule inhibitors of cyclin-dependent kinases (CDKs) are known to induce cell cycle arrest and apoptosis in certain cancer cells. In order to evaluate the antitumor activity of one such inhibitor, GW8510, against human lung cancers, we analyzed the effects of GW8510 on six nonsmall cell lung cancer (NSCLC) cell lines (A549, H1299, H460, H226, H358 and H322) and normal human fibroblast (NHFB). We treated the cells with GW8510 at concentrations of 0-10 microM, and found that it suppressed cell growth in vitro in all the lung cancer cells but not in NHFB. Subsequent study showed that GW8510 induced apoptosis and cell cycle arrest in the A549, H1299 and H460 cells in a time- and dose-dependent manner. Western blot analysis showed that GW8510 downregulated the expression of X-linked inhibitor of apoptosis (XIAP) but had no detectable effect on the expression of Bax, Bak, or Bcl2. GW8510 also downregulated XIAP mRNA level, suggesting that downregulation of XIAP expression occurs at the transcriptional level. Moreover, ectopic XIAP expression diminished growth inhibition and apoptosis induction by GW8510. Importantly, GW8510 was not capable of inducing apoptosis of NHFB cells. These results suggest that GW8510 might provide a treatment strategy for human NSCLC and XIAP is an important target for GW8510-induced apoptosis of NSCLC cells that occurs through inhibition of XIAP mRNA transcription.

Induction of apoptosis and down-regulation of Bcl-XL in cancer cells by a novel small molecule, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol.

In a search for new anticancer agents, we identified that 2[[3-(2,3-dichlorophenoxy) propyl]amino]ethanol (2,3-DCPE) induced apoptosis more effectively in various cancer cells than in normal human fibroblasts. We further evaluated the cell-killing effects of this compound in vitro in several human cancer cell lines and normal human fibroblasts. A cell viability assay showed that IC(50)s for human colon cancer cell lines LoVo and DLD-1, for human lung cancer cell lines H1299 and A549, and for normal human fibroblasts were 0.89, 1.95, 2.24, 2.69, and 12.6 micro M, respectively. Subsequent studies revealed that 2,3-DCPE could cause cleavage of caspase-8, caspase-3, caspase-9, and poly(ADP-ribose) polymerase and release of cytochrome c in cancer cells but not in normal human fibroblasts. Our data also showed that 2,3-DCPE attenuated the protein level of Bcl-XL and that apoptosis induction by 2,3-DCPE could be blocked by enforced overexpression of Bcl-XL. Our results suggest that 2,3-DCPE might be a potential new anticancer agent.

Bcl-XL small interfering RNA suppresses the proliferation of 5-fluorouracil-resistant human colon cancer cells.

5-Fluorouracil (5-FU) is commonly used to treat human colon cancers but resistance to this compound is frequently observed in clinics. To characterize mechanisms of resistance to 5-FU and to develop new strategies for overcoming it, we established two cell lines that were resistant to 5-FU but not other chemotherapeutic agents from parental 5-FU-sensitive cell lines. Western blot analysis revealed that these resistant cells overexpressed the proteins Bcl-XL, Bcl-Xs, and Bik, and further data showed that the cells were resistant to 5-FU-induced DNA damage and cell cycle disorder. However, in parental cells, enforced expression of Bcl-XL protein provided only limited protection from 5-FU-induced apoptosis and overexpression of Bcl-XL protein did not affect 5-FU-induced DNA damage or cell cycle changes; these findings suggested that overexpression of Bcl-XL protein was not the major contributor to 5-FU resistance in any of our cells lines. Even so, knockdown of Bcl-XL protein expression by Bcl-XL-specific small interfering RNA could inhibit proliferation more effectively in 5-FU-resistant cells than in 5-FU-sensitive cells, and the combination of Bcl-XL-specific small interfering RNA and 5-FU had additive effect on the inhibition of 5-FU-resistant cells. These results suggest that down-regulation of Bcl-XL protein expression might provide a new treatment strategy for human 5-FU-resistant colon cancer therapy.

Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino]ethanol in correlation with activation of ERK.

We recently found that a small molecule 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE) could induce apoptosis and downregulate Bcl-XL expression in various cancer cells. Here, we found that 2,3-DCPE suppressed the proliferation of Bcl-XL-overexpressing cancer cells without inducing apoptosis. Subsequently, we found that 2,3-DCPE could induce S-phase arrest and upregulate p21 but not p27 at a time- and dose-dependent but p53-dispensable manner in DLD-1 human colon cancer cells. Activation of ERK was also detected after treatment with 2,3-DCPE. Moreover, p21 induction was dramatically attenuated by ERK inhibitors PD98059 and U0126. Induction of p21 and S-phase arrest and corresponding activation of ERK were also observed in ATM-defective cells, suggesting that 2,3-DCPE-induced these events were ATM-dispensable. Furthermore, ERK inhibitors dramatically attenuated 2,3-DCPE-induced S-phase arrest. Together, our data indicate that ERK activation correlated with the 2,3-DCPE-mediated induction of p21 expression and S-phase arrest. This finding may have implication for cancer therapy.

Accelerated degradation of caspase-8 protein correlates with TRAIL resistance in a DLD1 human colon cancer cell line.

The tumor-selective cytotoxic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) makes TRAIL an attractive candidate as an anticancer agent. However, resistance to TRAIL poses a challenge in anticancer therapy with TRAIL. Therefore, characterizing the mechanisms of resistance and developing strategies to overcome the resistance are important steps toward successful TRAIL-mediated cancer therapy. In this study, we investigated mechanisms of acquired TRAIL resistance in a colon cancer DLD1 cell line. Compared with the TRAIL-susceptible DLD1 cell line, TRAIL-resistant DLD1/TRAIL-R cells have a low level of caspase-8 protein, but not its mRNA. Suppression of caspase-8 expression by siRNA in parental DLD1 cells led to TRAIL resistance. Restoration of caspase-8 protein expression by stable transfection rendered the DLD1/TRAIL-R cell line fully sensitive to TRAIL protein, suggesting that the low level of caspase-8 protein expression might be the culprit in TRAIL resistance in DLD1/TRAIL-R cells. Sequencing analysis of the caspase-8 coding region revealed a missense mutation that is present in both TRAIL-sensitive and TRAIL-resistant DLD1 cells. Subsequent study showed that the degradation of caspase-8 protein was accelerated in DLD1/TRAIL-R cells compared to parental DLD1 cells. Thus, accelerated degradation of caspase-8 protein is one of the mechanisms that lead to TRAIL resistance.

Activation of c-Jun NH2-terminal kinase is required for gemcitabine's cytotoxic effect in human lung cancer H1299 cells.

Although gemcitabine is a potent therapeutic agent in the treatment of human non-small cell lung cancer (NSCLC), resistance to gemcitabine is common. In this study, we investigated the molecular mechanisms involved in acquired gemcitabine resistance against NSCLC cells. Gemcitabine-resistant NSCLC H1299 cells (H1299/GR) were selected by long-term exposure of parental H1299 cells to gemcitabine. The median inhibitory concentrations of gemcitabine in H1299 and H1299/GR cells were 19.4 and 233.1 nM, respectively. Gemcitabine induced activation of c-Jun NH2-terminal kinase (JNK) in parental H1299 cells but not in H1299/GR cells after 48 h. Blocking JNK activation by pretreatment with SP600125, a specific JNK inhibitor, or by transfection with dominant-negative JNK vectors abrogated gemcitabine-induced apoptosis in parental H1299 cells as evidenced by interruption of caspase activation. Transient transfection with a JNKK2-JNK1 plasmid expressing constitutive JNK1 partially restored the effect of gemcitabine in H1299/GR cells. Our results indicate that gemcitabine-induced apoptosis in human NSCLC H1299 cells requires activation of the JNK signaling pathway. Attenuated JNK activation may contribute to development of acquired gemcitabine resistance in cancer cells.

Suppression of pancreatic tumor growth in the liver by systemic administration of the TRAIL gene driven by the hTERT promoter.

Local and locoregional administration of adenovectors expressing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been demonstrated to be useful in treating established tumors in animals. Moreover, expression of the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter can be used to prevent possible liver toxicity of the TRAIL gene. However, it remains unknown whether systemic administration of the TRAIL-expressing adenovector can be used for cancer therapy. Here, we showed that a combination of TRAIL gene therapy and gemcitabine, the first-line chemotheraphy agent for pancreatic cancer, had a synergistic effect on the induction of apoptosis in human pancreatic cancer cell lines in vitro. Systemic administration of an adenovector that contains an insertion of integrin-binding motif argine-glycine-aspartate (RGD) in the HI loop of the adenoviral fiber protein and expresses the human TRAIL gene from the hTERT promoter (designated Ad/TRAIL-F/RGD) suppressed the growth of human pancreatic tumor cells inoculated in the liver of nu/nu nude mice. Furthermore, Ad/TRAIL-F/RGD in combination with gemcitabine suppressed the tumor growth of pancreatic cancer in the liver more than did treatments consisting of each agent alone. No obvious liver toxicity was detected in any of the treatment groups. Our results suggest that TRAIL gene therapy in combination with gemcitabine might be a useful therapeutic approach for treating metastatic pancreatic cancers.

Proteasome inhibitors-mediated TRAIL resensitization and Bik accumulation.

Proteasome inhibitors can resensitize cells that are resistant to tumor necrosis factor-related apoptotic-inducing ligand (TRAIL)-mediated apoptosis. However, the underlying mechanisms of this effect are unclear. To characterize the mechanisms of interaction between proteasome inhibitors and TRAIL protein, we evaluated the effects of combined treatment with the proteasome inhibitors bortezomib and MG132 and TRAIL protein on two TRAIL-resistant human colon cancer cell lines, DLD1-TRAIL/R and LOVO-TRAIL/R. Both bortezomib and MG132 in combination with TRAIL enhanced apoptotosis induction in these cells, as evidenced by enhanced cleavage of caspases 8, 9, and 3, Bid, poly(ADP-ribose) polymerase and by the release of cytochrome C and Smac. Subsequent studies showed that combined treatment with bortezomib or MG132 resulted in an increase of death receptor (DR) 5 and Bik at protein levels but had no effects on protein levels of DR4, Bax, Bak, Bcl-2, Bcl-XL or Flice-inhibitory protein (FLIP). Moreover, c-Jun N-terminal kinase (JNK) is activated by these proteasome inhibitors. Blocking JNK activation with the JNK inhibitor SP600125 attenuated DR5 increase, but enhancement of apoptosis induction and increase of Bik protein were not affected. However, bortezomib-mediated TRAIL sensitization was partially blocked by using siRNA to knockdown Bik. Thus, our data suggests that accumulation of Bik may be critical for proteasome inhibitor-mediated resensitization of TRAIL.

Enhancing TRAIL-induced apoptosis by Bcl-X(L) siRNA.

We previously found that a change in the balance between mitochondrial pro- and anti-apoptotic proteins caused by ectopic expression of the Bax gene led to increased induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To investigate whether a similar effect can be elicited by down-regulating Bcl-X(L), an anti-apoptotic protein, we tested the effects of a small interfering RNA (siRNA) specific for Bcl-X(L) in TRAIL-resistant cells. The down-regulation of Bcl-X(L) by siRNA inhibited cell proliferation and sensitized TRAIL-induced apoptosis in human cancer cells with both acquired and intrinsic TRAIL resistance. Combining the Bcl-X(L) siRNA with TRAIL protein treatment resulted in an increase in the percentage of apoptotic cells and increased cleavage of caspase-8, caspase-9, caspase-3 and PARP. Furthermore, the release of cytochrome c but not Smac from mitochondria was induced by Bcl-X(L) siRNA alone, and this release was dramatically amplified by combining the Bcl-X(L) siRNA and TRAIL protein treatment. Together, our data suggest that simultaneous triggering of the death receptor and mitochondrial apoptotic pathways leads to enhanced induction of apoptosis, which makes it potentially useful for the treatment of resistant cancers.

Lack of p38 MAP kinase activation in TRAIL-resistant cells is not related to the resistance to TRAIL-mediated cell death.

Activation of MAP kinases is involved in various cellular processes, including immunoregulation, inflammation, cell growth, cell differentiation, and cell death. To investigate the role of p38 MAP kinase activation in the signaling pathway of TRAIL-mediated apoptosis, we compared TRAIL-mediated MAP kinase activation in TRAIL-susceptible human colon cancer cell line DLD1 and TRAIL-resistant DLD1/TRAIL-R cells. TRAIL-mediated activation of ERK occurred in both cell lines. In contrast, both DLD1 and DLD1/TRAIL-R cells showed no obvious JNK activation after treatment with TRAIL. Interestingly, TRAIL-mediated activation of p38 MAP kinases was observed in DLD1 cells but not in DLD1/TRAIL-R cells. However, activation of p38 MAP kinases was observed in both DLD1 and DLD1/TRAIL-R cells after treatment with anisomycin. Furthermore, inhibiting activated p38 MAP kinases with known inhibitors or with an adenovector expressing dominant negative p38alpha did not block TRAIL-mediated cell death in DLD1 cells. Moreover, activation of p38 MAP kinases by adenovectors expressing constitutive MKK3 or MKK6 (Ad/MKK3bE or Ad/MKK6bE) did not induce cell death in either DLD1 or DLD1/TRAIL-R cell lines. Our results suggest that activation of p38 MAP kinases does not play a major role in TRAIL-mediated apoptosis in DLD1 cells and that lack of TRAIL-mediated p38 MAP kinase activation may not be the mechanism of TRAIL-resistance in DLD1/TRAIL-R cells.