Targeting colorectal cancer stem cells with inducible caspase-9

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133+ CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo

Role of Bax in resveratrol-induced apoptosis of colorectal carcinoma cells

BACKGROUND: The natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with heart diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis, the latter possibly through the activation of pro-apoptotic proteins such as Bax. METHODS: The expression, subcellular localization, and importance of Bax for resveratrol-provoked apoptosis were assessed in human HCT116 colon carcinoma cells and derivatives with both bax alleles inactivated. RESULTS: Low to moderate concentrations of resveratrol induced co-localization of cellular Bax protein with mitochondria, collapse of the mitochondrial membrane potential, activation of caspases 3 and 9, and finally, apoptosis. In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent. Resveratrol inhibited the formation of colonies by both HCT116 and HCT116 bax -/- cells. CONCLUSION: Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis. Both can limit the ability of the cells to form colonies

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3.

Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the iron-sulfur cluster assembly (ISCU) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced ISCU in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect ISCU splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated ISCU. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of ISCU splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the ISCU transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced ISCU, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant ISCU and may, at least partially, explain the muscle-specific symptoms of HML

Incorrect splicing of <i>ISCU</i> in myoblasts with decreased SRSF3 expression.

<p>A lentivirus-mediated expression vector for SRSF3 shRNA (sh3) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) and myoblasts infected with a shSRSF3 lentivirus-mediated expression vector. The graph present the mean fold change ± SD for the SRSF3 expression from at least three independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 in non-transduced and transduced myoblasts. ACTIN was used as a loading reference. C) Semi-qRTPCR of human <i>ISCU</i> with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts, (-) or myoblasts infected with lentivirus-mediated vectors expressing shSRSF3 (sh3). D) Quantification of incorrectly spliced <i>ISCU</i> by qRTPCR in in non-transduced and transduced myoblasts. The graph presents the mean percentage of incorrectly spliced <i>ISCU</i> ± SD from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test).</p

Tissue-specific splicing of the human <i>ISCU</i> transgene.

<p>A) Schematic representation of the human <i>ISCU</i> transgene. Arrows mark the positions of primers used for semi-qRTPCR. The forward primer, present both in human and mouse, is located in exon 3. The reverse primers, specific to either human or mouse, are located in the 5’ UTR sequence in exon 5. The black thick line represents the last intron of the gene, which includes the pseudoexon represented by the black box. B) Semi-qRTPCR performed on cDNA from the human <i>ISCU</i> transgene and analyzed on a 1.2% agarose gel. Upper panel, human <i>ISCU</i> with incorrect (MUT) and correct (WT) splice variants. Lower panel, mouse endogenous <i>ISCU</i>. C) Quantification of the semi-qRTPCR assays using a 3730X DNA fragment analyzer (Applied Biosystems, Waltham, MA, USA). The graph shows the mean relative proportion ± SD of the splice variants, the wildtype (WT) and the two incorrect splice variants in which either 86 (MUT 86) or 100 bp (MUT 100) of intronic sequence (n = 10–14). D) qRTPCR performed on the mouse tissues (n = 6–10). The graph represents relative proportion incorrectly spliced <i>ISCU</i> (100bp). Tissues used were; muscle (M), heart (H), brain (B) liver (M) and kidney (K). All semi-qRTPCR and qRTPCR experiments where run in triplicates. (* p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test).</p

SRSF3 binding to normal and mutated <i>ISCU</i> RNA.

<p>A) SRSF3 qRTPCR using cDNA from patient (P1, P2) and control (C) myoblasts. The graph presents the mean fold expression ± SD in HML patient samples relative to the control from at least four independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 and GAPDH in HML patient myoblasts (P1, P2) and a healthy control (C). C) SRSF3 Western blot of the elute fractions (ELUTE) from RNA pull-down assays using patient (P1) and control (C) myoblasts. NE represents input nuclear extract. Nuclear extract was incubated without (-) or with biotinylated RNA oligos; <i>ISCU</i> wildtype oligo (WT), <i>ISCU</i> mutant oligo (MUT), consensus SRSF3 oligo (SRSF3) or scrambled RNA oligo (SCR). GAPDH was used as a negative control for RNA/protein interaction. The pull-down results were reproduced at least once for each oligo. D) Sequence of the <i>ISCU</i> RNA oligos used in the pull-down experiments with proposed SRSF3 binding sites indicated by horizontal square brackets. <b>C/G</b> indicates site of HML mutation. Start of the HML pseudoexon is boxed.</p

The High Level of Aberrant Splicing of <i>ISCU</i> in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3

<div><p>Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the <i>iron-sulfur cluster assembly</i> (<i>ISCU</i>) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced <i>ISCU</i> in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect <i>ISCU</i> splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated <i>ISCU</i>. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of <i>ISCU</i> splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the <i>ISCU</i> transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced <i>ISCU</i>, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant <i>ISCU</i> and may, at least partially, explain the muscle-specific symptoms of HML.</p></div