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.

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.

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.

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.

Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development.

Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/-) mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/-) mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/-) mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.

Redirecting adaptive immunity against foreign antigens to tumors for cancer therapy.

Immunotherapy for cancer is often limited by weak immunogenicity of tumor antigens. However, immune systems are usually strong and effective against foreign invading antigens. To test whether the destructive effect of adaptive immunity against foreign antigens can be redirected to tumors for cancer therapy, we immunized mice with adenovector expressing LacZ (Ad/CMV-LacZ). Subcutaneous syngeneic tumors were then established in the immunized animals or in naïve animals. The immune response against adenovirus or LacZ was redirected to tumors by intratumoral injection of Ad/CMV-LacZ. We found that immunization and treatment with the adenovector dramatically reduced the tumor growth rate compared with intratumoral administration of adenovector in naïve mice. Complete tumor regression was observed in about 50% of the immunized animals but not in the naïve animals. Similar effects were observed when oncolytic vaccinia virus was used to immunize and treat tumors. Lymphocyte infiltration in tumors was dramatically increased in the immunized group when compared with other groups. Moreover, immunity against parental tumor cells was induced in the animals cured with immunization and treatment with Ad/CMV-LacZ, as evidenced by the lack of tumor growth when the mice were challenged with parental tumor cells. Taken together, these results suggest that redirecting adaptive immunity against foreign antigens is a potential approach for anticancer therapy and that pre-existing immunity could enhance virotherapy against cancers.

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.

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.

A rapid and efficient method for purification of recombinant adenovirus with arginine-glycine-aspartic acid-modified fibers.

Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and size exclusion column chromatography. The purity and infectious activity of adenovectors isolated by the two methods were comparable. The new method yielded three to four times more adenovectors with arginine-glycine-aspartic acid (RGD)-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3h. Therefore, this method is rapid and efficient for production of recombination adenovectors, especially those with RGD-modified fibers.

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.

Antitumor activity and downregulation of pro-angiogenic molecules in human prostate cancer cells by a novel thiazolidione compound.

BACKGROUND: Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines. METHODS: The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay. RESULTS: DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 microM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G(2)/M phase and increased levels of G(2)/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H(3), and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 alpha and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS: DBPT can suppress proliferation, induce apoptosis, and down regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.

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.

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.

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.

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.

P-glycoprotein-independent apoptosis induction by a novel synthetic compound, MMPT [5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone].

To develop new anticancer agents that are effective for treatment of chemoresistant tumors, we screened a chemical library for compounds that can effectively kill both paclitaxel-sensitive lung cancer cell H460 and P-glycoprotein-overexpressing paclitaxel-resistant cell H460/TaxR. A synthetic compound, MMPT (5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone), was identified to induce cytotoxic effects in both H460 and H460/TaxR cells but not in normal fibroblasts. MMPT effectively inhibited the growth of several human lung cancer cell lines in a dose-dependent manner, with 50% inhibitory concentrations ranging from 4.9 to 8.0 microM. The inhibitory effect on cancer cells is independent of the status of p53 and P-glycoprotein. Moreover, MMPT had no obvious toxic effects on normal human fibroblasts and mesenchymal stem cells at the 50% inhibitory concentration for lung cancer cell lines. Treating lung cancer cells with MMPT-induced apoptosis with caspase-3, -8, -9, and poly(ADP-ribose) polymerase cleavage and cytochrome c release from mitochondria. MMPT-induced apoptosis was abrogated when c-Jun N-terminal kinase (JNK) activation was blocked with a specific JNK inhibitor, SP600125. Furthermore, in vivo administration of MMPT suppressed human H460 xenograft tumor growth in nude mice. Our results suggest that MMPT may induce tumor-selective cell killing in both P-glycoprotein-negative and -positive cancer cells and could be a new anticancer agent for treatment of refractory tumors.

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.

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.