Modeling scalability of impurity precipitation in downstream biomanufacturing.

Precipitation during the viral inactivation, neutralization and depth filtration step of a monoclonal antibody (mAb) purification process can provide quantifiable and potentially significant impurity reduction. However, robust commercial implementation of this unit operation is limited due to the lack of a representative scale-down model to characterize the removal of impurities. The objective of this work is to compare isoelectric impurity precipitation behavior for a monoclonal antibody product across scales, from benchtop to pilot manufacturing. Scaling parameters such as agitation and vessel geometry were investigated, with the precipitate amount and particle size distribution (PSD) characterized via turbidity and flow imaging microscopy. Qualitative analysis of the data shows that maintaining a consistent energy dissipation rate (EDR) could be used for approximate scaling of vessel geometry and agitator speeds in the absence of more detailed simulation. For a more rigorous approach, however, agitation was simulated via computational fluid dynamics (CFD) and these results were applied alongside a population balance model to simulate the trajectory of the size distribution of precipitate. CFD results were analyzed within a framework of a two-compartment mixing model comprising regions of high- and low-energy agitation, with material exchange between the two. Rate terms accounting for particle formation, growth and breakage within each region were defined, accounting for dependence on turbulence. This bifurcated model was successful in capturing the variability in particle sizes over time across scales. Such an approach enhances the mechanistic understanding of impurity precipitation and provides additional tools for model-assisted prediction for process scaling.

Newly reconstructed Arctic surface air temperatures for 1979-2021 with deep learning method.

A precise Arctic surface air temperature (SAT) dataset, that is regularly updated, has more complete spatial and temporal coverage, and is based on instrumental observations, is critically important for timely monitoring and improving understanding of the rapid change in the Arctic climate. In this study, a new monthly gridded Arctic SAT dataset dated back to 1979 was reconstructed with a deep learning method by combining surface air temperatures from multiple data sources. The source data include the observations from land station of GHCN (Global Historical Climatology Network), ICOADS (International Comprehensive Ocean-Atmosphere Data Set) over the oceans, drifting ice station of Russian NP (North Pole), and buoys of IABP (International Arctic Buoy Programme). The last two are crucial for improving the representation of the in-situ observed temperatures within the Arctic. The newly reconstructed dataset includes monthly Arctic SAT beginning in 1979 and daily Arctic SAT beginning in 2011. This dataset would represent a new improvement in developing observational temperature datasets and can be used for a variety of applications.

Effect of solution condition on the binding behaviors of monoclonal antibody and fusion protein therapeutics in Protein A chromatography.

Recent development in Protein A chromatography has adopted wash buffers with a wide range of pH to improve the removal of process-related impurities, e.g., host cell proteins (HCPs), from Fc-based target biological products. Thus, it is important to investigate the pH effect on the binding behaviors of target products of various properties on Protein A resins. Here we performed column breakthrough experiments for two monoclonal antibodies (mAbs) and one Fc-fusion protein on two Protein A resins from pH 4 to pH 10, and the experimental data was analyzed using a mechanistic model to obtain isotherm and mass transfer parameters. The two mAbs exhibited relatively high dynamic binding capacity (DBC) at 10% breakthrough of 43 - 67 g/L at pH 6-9 followed by a ∼30% decrease from pH 9 to pH 10; while the Fc-fusion protein reached the highest DBC at 10% breakthrough of 10 - 17 g/L at pH 5 and thereafter the capacity gradually reduced. Model analyses revealed that the two mAbs had higher maximum binding capacity by 2 - 5 fold but weaker binding affinity (12 - 64%) than the Fc-fusion protein from pH 5 to pH 9. For the three molecules, similar patterns of the pH impact were observed on the two Protein A resins with the Jetted A50 resin showing generally higher DBC and stronger binding affinity than the MabSelect SuRe LX resin. Additionally, an inverse relationship between the binding affinity and surface diffusivity was observed for both resins. Besides obtaining the isotherm parameters from the column breakthrough data, a direct batch equilibrium measurement showed comparable trend in these parameters with relatively more scattered values due to the inherent uncertainties to accurately determine the initial slope of the isotherm in highly favorable adsorption conditions. Finally, isothermal titration calorimetry (ITC) results revealed that the measured binding affinity using free Protein A ligand was stronger than that obtained from the breakthrough and isotherm results for the resin, possibly due to the reduced accessibility of the immobilized ligand on resin surface. Overall, this work can facilitate future Protein A ligand design and affinity chromatography process optimization for biomanufacturing.

Role of harvest depth filtration in controlling product-related impurities for a bispecific antibody.

Background: Bispecific antibodies (BsAb) belong to a novel antibody category with advantages over traditional mono-specific therapeutic antibodies. However, product variants are also commonly seen during the production of BsAb, which poses significant challenges to downstream processing. In this study, the adsorptive characteristics of a BsAb product and its variants were investigated for a set of depth filters during primary recovery of the cell culture fluid. Methods: The retention of the BsAb product and its variants on a set of Millistak+® D0HC and X0HC depth filters were first investigated, followed by studying the mechanism of their adsorption on the depth filters. The chemical and structural properties of depth filters along with the molecular properties of the product and its variants were studied subsequently. Results: The X0HC filter was found to be able to retain a significant amount of low molecular weight (LMW) variants along with a low amount of main product retained. Different levels of retention, observed for these variants, were correlated to their different hydrophobic and charge characteristics in relation with the adsorptive properties of the depth filters used. Electrostatic, hydrophobic, and hydrogen bonding interactions were found to be the key forces to keep product variants retained on the depth filter where the higher hydrophobicity of the LMW variants may cause them to be preferentially retained. Conclusion: Harvest depth filters potentially can be utilized for retaining the BsAb variants, which depends on relative molecular properties of the product and its variants and adsorptive properties of the depth filters used.

Fruit quality prediction based on soil mineral element content in peach orchard.

Mineral nutrition of orchard soil is critical for the growth of fruit trees and improvement of fruit quality. In the present study, the effects of soil mineral nutrients on peach fruit quality were studied by using artificial neural network model. The results showed that the four established ANN models had the highest prediction accuracy (R 2 = .9735, .9607, .9036, and .9440, respectively). The results of prediction model sensitivity analysis showed that available B, Ca, N, and K in the soil had the greatest influence on the single fruit weight, available Fe, K, B, and Ca in the soil had the greatest effect on fruit soluble solid content, available Ca, N, B, and K in the soil had the greatest influence on the fruit titratable acid content, and available Ca, Fe, N, and Mn in the soil had the greatest effect on fruit edible rate. The response surface methodology analysis determined the optimal range of these mineral elements, which is critical for guiding precision fertilization in peach orchards and improving peach fruit quality.

Molecular and Biological Characterization of Two New Species Causing Peach Shoot Blight in China.

Peach shoot blight (PSB), which kills shoots, newly sprouted leaf buds, and peach fruits, has gradually increased over the last 10 years and resulted in 30 to 50% of total production loss of the peach industry in China. Phomopsis amygdali has been identified as the common causal agent of this disease. In this study, two new species, Phomopsis liquidambaris (strain JW18-2) and Diaporthe eres (strain JH18-2), were also pathogens causing PSB, as determined through molecular phylogenetic analysis based on the sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α) and beta-tubulin (TUB), and colony and conidial morphological characteristics. Biological phenotypic analysis showed that the colony growth rate of strain JW18-2 was faster than that of strains JH18-2 and ZN32 (one of the P. amygdali strains that we previously found and identified). All three strains produced α-conidia; however, JW18-2 could not produce ß-conidia on alfalfa decoction and Czapek media, and the ß-conidia produced by strain JH18-2 were shorter in length and thicker in width than those produced by strain ZN32. Pathogenicity tests showed that JW18-2 presented the strongest pathogenicity for peach fruits and twigs and was followed by strains JH18-2 and ZN32. The results shed light on the etiology of PSB and provide a warning that P. liquidambaris or D. eres might develop into dominant species after a few years while also potentially benefitting the development of effective disease control management strategies.

First report of Geotrichum candidum causing sour rot of peach in China.

Peach (Prunus persica L. Batsch) is one of the most important fruit crops in China (Wang et al. 2011). Yangshan Town of Jiangsu Province is one of the four major peach producing areas in China, with a growing area of 2,000 ha (Tian et al. 2018). During June 2020, a postharvest disease presenting with brown necrosis and rot occurred on peaches in Yangshan Town. The estimated damage was more than 10% of the total harvest. The symptoms included soft rot, and the lesion appeared sunken, accompanied with sour odor and white mycelia. Twelve peaches with representative symptom were sampled for pathogen isolation. Pieces (about 5 mm × 5 mm) from the lesion edge of symptomatic fruits were dissected and surface disinfected (3% NaClO for 10 s and 75% ethanol for 30 s), then rinsed three times with distilled water, dried on sterile filter paper and transferred to Potato Dextrose Agar (PDA) media plates supplemented with 150 ng/mL streptomycin sulfate. The plates were incubated at 28 ℃ for 3 days. Forty-eight isolations were obtained from the plates and isolates were single-spored. All isolates presented white, flat, milky yeast-like colonies with radial mycelia. Hyphae under microscope were septate, branched, disarticulating into arthroconidia measuring 3.39 to 9.27 × 2.05 to 7.71 µm. The morphological characteristics are consistent with Geotrichum candidum (De Hoog et al. 1986). Internal transcribed spacer (ITS) and 18s nuclear ribosomal small subunit (SSU) of the 48 isolates were amplified and sequenced using the primers ITS5/ITS4, and NS1/NS4 for molecular identification (Schoch et al. 2012). The resulted sequences showed no difference among all the isolates. Alignment by blastn showed the sequence of ITS and SSU were 100% (accession number. GQ376093) and 99.7% identical (accession number. KY977411.1) to Geotrichum candidum, respectively. The sequences of ITS (accession number MW493646) and SSU (accession number MW493648) were submitted to the GenBank. Commercial ripe peaches with the size of about 15 cm × 15 cm × 10 cm was used for pathogenicity test. Peaches were surface disinfected with 75% ethanol, then a wound with 4 mm in diameter and 5 mm in depth was made on the surface of each fruit. Ten peaches were inoculated with 10 µL (1×105 spores /mL) of the isolate suspension. Another ten peaches were inoculated with 10 µL sterile water as the control. Peaches were incubated individually at 28 ℃and a relative humidity of about 85%. After three days, large scale of pits and necrosis appeared on every peach inoculated, and the symptoms were consistent with the diseased peaches in Yangshan Town, while no symptoms non-inoculated on the control peaches were observed. The pathogen was re-isolated from the diseased fruit and was identified again by sequencing of ITS and SSU. All the tests were conducted three times. Considering the evidence, we identified the pathogen as G. candidum. This pathogen has been reported to cause sour rot was reported in kiwifruit, strawberry, melon and other fruits (Alonzo et al. 2020; Cheng et al. 2020; Halfeld-Vieira et al. 2020). To our knowledge, this is the first report of G. candidum causing sour rot of peach in China, which may cause a great loss to peach industry of China.

Productivity improvement and charge variant modulation for intensified cell culture processes by adding a carboxypeptidase B (CpB) treatment step.

The goal of cell culture process intensification is to improve productivity while maintaining acceptable quality attributes. In this report, four processes, namely a conventional manufacturing Process A, and processes intensified by enriched N-1 seed (Process B), by perfusion N-1 seed (Process C), and by perfusion production (Process D) were developed for the production of a monoclonal antibody. The three intensified processes substantially improved productivity, however, the product either failed to meet the specification for charge variant species (main peak) for Process D or the production process required early harvest to meet the specification for charge variant species, Day 10 or Day 6 for Processes B and C, respectively. The lower main peak for the intensified processes was due to higher basic species resulting from higher C-terminal lysine. To resolve this product quality issue, we developed an enzyme treatment method by introducing carboxypeptidase B (CpB) to clip the C-terminal lysine, leading to significantly increased main peak and an acceptable and more homogenous product quality for all the intensified processes. Additionally, Processes B and C with CpB treatment extended bioreactor durations to Day 14 increasing titer by 38% and 108%, respectively. This simple yet effective enzyme treatment strategy could be applicable to other processes that have similar product quality issues.

Hydrophobic property of cation-exchange resins affects monoclonal antibody aggregation.

High molecular weight (HMW) aggregate formation of therapeutic monoclonal antibodies (mAbs) during cation-exchange chromatography (CEX) has been frequently observed, and can be a challenge for downstream purification. To gain mechanistic understanding of this phenomenon, aggregate formation in bind-elute CEX for two therapeutic mAbs (IgG1 and IgG4) was examined on three CEX resins (Capto SP ImpRes, Fractogel EMD SE Hicap, and POROS XS). First, mAb structural stability was studied in solutions under CEX load conditions. Using differential scanning fluorimetry (DSF), the measured melting temperature (Tm DSF (Unbound)) decreased from 60.7 to 52.4°C for mAb1 and 51.5 to 45.2°C for mAb2 when lowering pH from 6.0 to 4.5. Then, mAb structural stability was further investigated in the bound state on CEX surfaces. Using differential scanning calorimetry (DSC), the measured melting temperature of the bound mAbs (Tm DSC (Bound)) was 4.5 - 6.5°C lower than that for the unbound mAbs (Tm DSC (Unbound)) in the same solutions. The Tm differences (∆Tm DSC (Unbound minus Bound)) between the two states correlated with the severity of mAb aggregation in CEX operations, indicating the importance of both intrinsic mAb stability and resin properties. In particular, resin hydrophobicity was shown to have a critical impact. The interplay among these protein- and resin-related factors, together with solution conditions, ultimately dictates the aggregate formation observed. Finally, the hydrophobicity of the CEX resins (Capto SP ImpRes < Fractogel EMD SE Hicap < POROS XS) was measured using a fluorescence-based method to quantitatively characterize this resin property. Results suggest that the mAb-accessible hydrophobic regions of the CEX resins affect the structural stability of the bound mAbs to various degrees, leading to differences in aggregate formation upon mAb elution. In summary, this study offers insight into the mechanism of mAb aggregation in bind-elute CEX operations, and the in-depth understanding facilitates the development of robust CEX conditions for mAb purification.

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production.

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.

Protein aggregation and mitigation strategy in low pH viral inactivation for monoclonal antibody purification.

Significant amounts of soluble product aggregates were observed during low-pH viral inactivation (VI) scale-up for an IgG4 monoclonal antibody (mAb IgG4-N1), while small-scale experiments in the same condition showed negligible aggregation. Poor mixing and product exposure to low pH were identified as the root cause. To gain a mechanistic understanding of the problem, protein aggregation properties were studied by varying critical parameters including pH, hold time and protein concentration. Comprehensive biophysical characterization of product monomers and aggregates was performed using fluorescence-size-exclusion chromatography, differential scanning fluorimetry, fluorescence spectroscopy, and dynamic light scattering. Results showed IgG4-N1 partially unfolds at about pH 3.3 where the product molecules still exist largely as monomers owing to strong inter-molecular repulsions and favorable colloidal stability. In the subsequent neutralization step, however, the conformationally changed monomers are prone to aggregation due to weaker inter-molecular repulsions following the pH transition from 3.3 to 5.5. Surface charge calculations using homology modeling suggested that intra-molecular repulsions, especially between CH2 domains, may contribute to the IgG4-N1 unfolding at ≤ pH 3.3. Computational fluid dynamics (CFD) modeling was employed to simulate the conditions of pH titration to reduce the risk of aggregate formation. The low-pH zones during acid addition were characterized using CFD modeling and correlated to the condition causing severe product aggregation. The CFD tool integrated with the mAb solution properties was used to optimize the VI operating parameters for successful scale-up demonstration. Our research revealed the governing aggregation mechanism for IgG4-N1 under acidic conditions by linking its molecular properties and various process-related parameters to macroscopic aggregation phenomena. This study also provides useful insights into the cause and mitigation of low-pH-induced IgG4 aggregation in downstream VI operation.

Lappaol F, a novel anticancer agent isolated from plant arctium Lappa L.

In an effort to search for new cancer-fighting therapeutics, we identified a novel anticancer constituent, Lappaol F, from plant Arctium Lappa L. Lappaol F suppressed cancer cell growth in a time- and dose-dependent manner in human cancer cell lines of various tissue types. We found that Lappaol F induced G(1) and G(2) cell-cycle arrest, which was associated with strong induction of p21 and p27 and reduction of cyclin B1 and cyclin-dependent kinase 1 (CDK1). Depletion of p21 via genetic knockout or short hairpin RNA (shRNA) approaches significantly abrogated Lappaol F-mediated G(2) arrest and CDK1 and cyclin B1 suppression. These results suggest that p21 seems to play a crucial role in Lappaol F-mediated regulation of CDK1 and cyclin B1 and G(2) arrest. Lappaol F-mediated p21 induction was found to occur at the mRNA level and involved p21 promoter activation. Lappaol F was also found to induce cell death in several cancer cell lines and to activate caspases. In contrast with its strong growth inhibitory effects on tumor cells, Lappaol F had minimal cytotoxic effects on nontumorigenic epithelial cells tested. Importantly, our data also demonstrate that Lappaol F exhibited strong growth inhibition of xenograft tumors in nude mice. Lappaol F was well tolerated in treated animals without significant toxicity. Taken together, our results, for the first time, demonstrate that Lappaol F exhibits antitumor activity in vitro and in vivo and has strong potential to be developed as an anticancer therapeutic.

[Diagnosis and treatment of cerebral schistosomiasis: a report of 166 cases].

OBJECTIVE: To discuss the clinical features, diagnosis and treatment of cerebral schistosomiasis. METHODS: A total of 166 patients with cerebral schistosomiasis were treated, and their clinical data were collected and analyzed retrospectively. RESULTS: In 166 cases of cerebral schistosomiasis, the confirmative diagnoses of 156 cases were diagnosed according to the clinical manifestation, etiology, immunology and auxiliary examination (CT, MRI). In among, 74 cases were confirmed by pathological examination, 10 cases were diagnosed through to the diagnostic treatment. Totally 102 patients received the oral medication of praziquantel, and they all improved and discharged 14-16 days later; 64 patients received the craniotomy and praziquantel medication after the operation, and 48 patients significantly improved, others did not improve or aggravated. There was no operative mortality. CONCLUSIONS: Neuroimaging and laboratory tests are valuable in the diagnosis of cerebral schistosomiasis. The praziquantel treatment is selected firstly when the diagnosis was established. However, in the case of serious intracranial hypertension, intractable epilepsy and praziquantel treatment fails, the surgical treatment is required.

DOC45, a novel DNA damage-regulated nucleocytoplasmic ATPase that is overexpressed in multiple human malignancies.

In this article, we report the characterization of a novel DNA damage-regulated gene, named DNA damage-regulated overexpressed in cancer 45 (DOC45). Our results indicate that DNA damage-inducing agents, including doxorubicin (adriamycin), etoposide, and ionizing and UV radiation, strongly downregulate DOC45 expression, whereas endoplasmic reticulum stress-inducing agents do not. Our results also indicate that DOC45 is overexpressed in several human malignancies, including cancers of the colon, rectum, ovary, lung, stomach, and uterus. DOC45 harbors conserved nucleotide triphosphate-binding motifs and is capable of ATP hydrolysis, findings that highlight its function as a novel ATPase. Although predominantly cytoplasmic, DOC45 exhibits a characteristic nucleocytoplasmic distribution and, on inhibition of nuclear export, predominantly accumulates in the nucleoli. These results suggest that DOC45 may shuttle between nucleus and cytoplasm to carry out its function. Our results also indicate that DOC45 expression is enhanced during oncogenic Ras-mediated transformation and that its expression is linked to phosphoinositide 3-kinase signaling pathway. Furthermore, short hairpin RNA-mediated knockdown of DOC45 in human colon cancer cells inhibits their proliferation and enhances cellular sensitivity to doxorubicin-induced cell death, suggesting that DOC45 plays an important role in cell proliferation and survival. Collectively, our results indicate that DOC45 is a novel ATPase that is linked to cellular stress response and tumorigenesis, and may also serve as a valuable tumor marker.

RBEL1 is a novel gene that encodes a nucleocytoplasmic Ras superfamily GTP-binding protein and is overexpressed in breast cancer.

Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.

Oncogenic Ras-mediated downregulation of Gadd153/CHOP is required for Ras-induced cellular transformation.

Oncogenic Ras proteins transform cells via multiple downstream signaling cascades that are important for cell proliferation and survival. Gadd153, also known as CHOP, is a growth inhibitory and proapoptotic protein and its expression is upregulated by many agents that induce apoptosis. Here, we report our novel findings that oncogenic Ras downregulates Gadd153 expression at both protein and mRNA levels and that such downregulation occurs, at least in part, via decreases in GADD153 mRNA stability. Gadd153 downregulation is specific to oncogenic Ras since another oncogenic family member R-Ras2/TC21 does not downregulate Gadd153. We further demonstrate that the expression of exogenous Gadd153 interferes with Ras-induced oncogenic transformation, which suggests that downregulation of Gadd153 appears to be an important mechanism by which oncogenic Ras promotes cellular transformation. Thus, oncogenic Ras-mediated cellular transformation also involves downmodulation of important molecules such as Gadd153 that negatively regulate cell growth and survival.

Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest.

RASSF1A is a putative tumor suppressor gene that is inactivated in a variety of human tumors. Expression of exogenous RASSF1A has been shown to inhibit tumor growth in vitro and in animals. However, the molecular mechanisms by which RASSF1A mediates its tumor suppressive effects remain to be elucidated. Here, we report that RASSF1A is a microtubule-binding protein that interacts with and stabilizes microtubules. We have identified the RASSF1A region harboring a basic domain that appears to mediate the interactions between RASSF1A and microtubules. The basic domain-containing RASSF1C isoform also interacts with and stabilizes microtubules. We further show that in addition to G1 arrest, RASSF1A promotes growth arrest in the G2/M phase of the cell cycle and endogenous RASSF1A also interacts with microtubules. Based on our results, we propose that RASSF1A may mediate its tumor suppressive effects by inducing growth arrest in the G1 and G2/M phases. Together, these results provide important new insights into the molecular mechanisms by which this novel tumor suppressor mediates its biological effects.

Effect of Bax deficiency on death receptor 5 and mitochondrial pathways during endoplasmic reticulum calcium pool depletion-induced apoptosis.

Thapsigargin (TG), by inducing perturbations in cellular Ca(2+) homeostasis, can induce apoptosis, but the molecular mechanisms remain to be fully elucidated. We have recently reported that TG-induced apoptosis appears to involve the DR5-dependent apoptotic pathway that cross talks with the mitochondrial pathway via TG-induced Bid cleavage. In this study, we have utilized Bax-proficient and -deficient HCT116 human colon cancer cells to investigate the effect of Bax deficiency on TG-induced apoptosis and TG regulation of the DR5 and mitochondrial pathways. Our results indicate that Bax-deficient cells are less sensitive to undergo apoptosis following TG treatment. Our results further demonstrate that TG-induced apoptosis is coupled with DR5 upregulation and caspases 8 and 3 activation, as well as Bid cleavage in both Bax-proficient and -deficient cells, although caspase 3 activation was reduced in Bax-deficient cells. TG also promoted the release of cytochrome c into cytosol and caspase 9 activation in Bax-proficient cells but not in Bax-deficient cells. These findings suggest that although Bax is not absolutely required for death receptor (DR)-dependent signals, it appears to be a key molecule in TG-regulated mitochondrial events. Bax-deficient cells were relatively more resistant to Apo2L/TRAIL than the Bax-proficient counterparts. However, the combination of Apo2L/TRAIL and TG was more effective in mediating apoptosis in both Bax-proficient and -deficient cells and that was coupled with activation of caspases 8 and 3. Although both agents in combination also induced cytochrome c release into cytosol and caspase 9 activation in Bax-proficient cells, these events were abrogated in Bax-deficient cells. Our results thus suggest that the combination of Apo2L/TRAIL and TG appears to bypass the Bax deficiency-induced defects in the mitochondrial (intrinsic) pathway by engaging the DR5-dependent apoptotic signals (extrinsic pathway).