Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells
Identifieur interne : 000431 ( Pmc/Curation ); précédent : 000430; suivant : 000432Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells
Auteurs : Marina Solovieva ; Yuri Shatalin ; Roman Fadeev ; Olga Krestinina ; Yulia Baburina ; Alexey Kruglov ; Ekaterina Kharechkina ; Margarita Kobyakova ; Vadim Rogachevsky ; Elena Shishkova ; Vladimir AkatovSource :
- Biomolecules [ 2218-273X ] ; 2020.
Abstract
We have shown that hydroxycobalamin (vitamin B12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + B12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.
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DOI: 10.3390/biom10010069
PubMed: 31906414
PubMed Central: 7023477
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Vitamin B<sub>12b</sub> Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells</title><author><name sortKey="Solovieva, Marina" sort="Solovieva, Marina" uniqKey="Solovieva M" first="Marina" last="Solovieva">Marina Solovieva</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Shatalin, Yuri" sort="Shatalin, Yuri" uniqKey="Shatalin Y" first="Yuri" last="Shatalin">Yuri Shatalin</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Fadeev, Roman" sort="Fadeev, Roman" uniqKey="Fadeev R" first="Roman" last="Fadeev">Roman Fadeev</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Krestinina, Olga" sort="Krestinina, Olga" uniqKey="Krestinina O" first="Olga" last="Krestinina">Olga Krestinina</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Baburina, Yulia" sort="Baburina, Yulia" uniqKey="Baburina Y" first="Yulia" last="Baburina">Yulia Baburina</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Kruglov, Alexey" sort="Kruglov, Alexey" uniqKey="Kruglov A" first="Alexey" last="Kruglov">Alexey Kruglov</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Kharechkina, Ekaterina" sort="Kharechkina, Ekaterina" uniqKey="Kharechkina E" first="Ekaterina" last="Kharechkina">Ekaterina Kharechkina</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Kobyakova, Margarita" sort="Kobyakova, Margarita" uniqKey="Kobyakova M" first="Margarita" last="Kobyakova">Margarita Kobyakova</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Rogachevsky, Vadim" sort="Rogachevsky, Vadim" uniqKey="Rogachevsky V" first="Vadim" last="Rogachevsky">Vadim Rogachevsky</name><affiliation><nlm:aff id="af2-biomolecules-10-00069">Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>vadim_rogachevsky@synapsis.ru</email> (V.R.);<email>shishkova@neuro.nnov.ru</email> (E.S.)</nlm:aff></affiliation></author><author><name sortKey="Shishkova, Elena" sort="Shishkova, Elena" uniqKey="Shishkova E" first="Elena" last="Shishkova">Elena Shishkova</name><affiliation><nlm:aff id="af2-biomolecules-10-00069">Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>vadim_rogachevsky@synapsis.ru</email> (V.R.);<email>shishkova@neuro.nnov.ru</email> (E.S.)</nlm:aff></affiliation></author><author><name sortKey="Akatov, Vladimir" sort="Akatov, Vladimir" uniqKey="Akatov V" first="Vladimir" last="Akatov">Vladimir Akatov</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31906414</idno><idno type="pmc">7023477</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7023477</idno><idno type="RBID">PMC:7023477</idno><idno type="doi">10.3390/biom10010069</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000431</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000431</idno><idno type="wicri:Area/Pmc/Curation">000431</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000431</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Vitamin B<sub>12b</sub> Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells</title><author><name sortKey="Solovieva, Marina" sort="Solovieva, Marina" uniqKey="Solovieva M" first="Marina" last="Solovieva">Marina Solovieva</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Shatalin, Yuri" sort="Shatalin, Yuri" uniqKey="Shatalin Y" first="Yuri" last="Shatalin">Yuri Shatalin</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Fadeev, Roman" sort="Fadeev, Roman" uniqKey="Fadeev R" first="Roman" last="Fadeev">Roman Fadeev</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Krestinina, Olga" sort="Krestinina, Olga" uniqKey="Krestinina O" first="Olga" last="Krestinina">Olga Krestinina</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Baburina, Yulia" sort="Baburina, Yulia" uniqKey="Baburina Y" first="Yulia" last="Baburina">Yulia Baburina</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Kruglov, Alexey" sort="Kruglov, Alexey" uniqKey="Kruglov A" first="Alexey" last="Kruglov">Alexey Kruglov</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Kharechkina, Ekaterina" sort="Kharechkina, Ekaterina" uniqKey="Kharechkina E" first="Ekaterina" last="Kharechkina">Ekaterina Kharechkina</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Kobyakova, Margarita" sort="Kobyakova, Margarita" uniqKey="Kobyakova M" first="Margarita" last="Kobyakova">Margarita Kobyakova</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author><author><name sortKey="Rogachevsky, Vadim" sort="Rogachevsky, Vadim" uniqKey="Rogachevsky V" first="Vadim" last="Rogachevsky">Vadim Rogachevsky</name><affiliation><nlm:aff id="af2-biomolecules-10-00069">Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>vadim_rogachevsky@synapsis.ru</email> (V.R.);<email>shishkova@neuro.nnov.ru</email> (E.S.)</nlm:aff></affiliation></author><author><name sortKey="Shishkova, Elena" sort="Shishkova, Elena" uniqKey="Shishkova E" first="Elena" last="Shishkova">Elena Shishkova</name><affiliation><nlm:aff id="af2-biomolecules-10-00069">Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>vadim_rogachevsky@synapsis.ru</email> (V.R.);<email>shishkova@neuro.nnov.ru</email> (E.S.)</nlm:aff></affiliation></author><author><name sortKey="Akatov, Vladimir" sort="Akatov, Vladimir" uniqKey="Akatov V" first="Vladimir" last="Akatov">Vladimir Akatov</name><affiliation><nlm:aff id="af1-biomolecules-10-00069">Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</nlm:aff></affiliation></author></analytic><series><title level="j">Biomolecules</title><idno type="eISSN">2218-273X</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>We have shown that hydroxycobalamin (vitamin B<sub>12b</sub>) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + B<sub>12b</sub>. It was found that cell death induced by DDC + B<sub>12b</sub> differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B<sub>12b</sub> used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Hendren, R L" uniqKey="Hendren R">R.L. Hendren</name></author><author><name sortKey="James, S J" uniqKey="James S">S.J. James</name></author><author><name sortKey="Widjaja, F" uniqKey="Widjaja F">F. Widjaja</name></author><author><name sortKey="Lawton, B" uniqKey="Lawton B">B. Lawton</name></author><author><name sortKey="Rosenblatt, A" uniqKey="Rosenblatt A">A. Rosenblatt</name></author><author><name sortKey="Bent, S" uniqKey="Bent S">S. Bent</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thompson, J P" uniqKey="Thompson J">J.P. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biomolecules</journal-id><journal-id journal-id-type="iso-abbrev">Biomolecules</journal-id><journal-id journal-id-type="publisher-id">biomolecules</journal-id><journal-title-group><journal-title>Biomolecules</journal-title></journal-title-group><issn pub-type="epub">2218-273X</issn><publisher><publisher-name>MDPI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31906414</article-id><article-id pub-id-type="pmc">7023477</article-id><article-id pub-id-type="doi">10.3390/biom10010069</article-id><article-id pub-id-type="publisher-id">biomolecules-10-00069</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Vitamin B<sub>12b</sub> Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Solovieva</surname><given-names>Marina</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><name><surname>Shatalin</surname><given-names>Yuri</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><name><surname>Fadeev</surname><given-names>Roman</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><name><surname>Krestinina</surname><given-names>Olga</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><name><surname>Baburina</surname><given-names>Yulia</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-9215-7387</contrib-id><name><surname>Kruglov</surname><given-names>Alexey</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><name><surname>Kharechkina</surname><given-names>Ekaterina</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><name><surname>Kobyakova</surname><given-names>Margarita</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-9611-419X</contrib-id><name><surname>Rogachevsky</surname><given-names>Vadim</given-names></name><xref ref-type="aff" rid="af2-biomolecules-10-00069">2</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0002-7402-8546</contrib-id><name><surname>Shishkova</surname><given-names>Elena</given-names></name><xref ref-type="aff" rid="af2-biomolecules-10-00069">2</xref></contrib><contrib contrib-type="author"><name><surname>Akatov</surname><given-names>Vladimir</given-names></name><xref ref-type="aff" rid="af1-biomolecules-10-00069">1</xref><xref rid="c1-biomolecules-10-00069" ref-type="corresp">*</xref></contrib></contrib-group><aff id="af1-biomolecules-10-00069"><label>1</label>Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>m_solovieva@iteb.ru</email> (M.S.);<email>yury.shatalin@yandex.ru</email> (Y.S.);<email>fadeevrs@gmail.com</email> (R.F.);<email>ovkres@mail.ru</email> (O.K.);<email>byul@rambler.ru</email> (Y.B.);<email>krugalex@rambler.ru</email> (A.K.);<email>katya.kypri@gmail.com</email> (E.K.);<email>ritaaaaa49@gmail.com</email> (M.K.)</aff><aff id="af2-biomolecules-10-00069"><label>2</label>Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia;<email>vadim_rogachevsky@synapsis.ru</email> (V.R.);<email>shishkova@neuro.nnov.ru</email> (E.S.)</aff><author-notes><corresp id="c1-biomolecules-10-00069"><label>*</label>Correspondence: <email>akatov.vladimir@gmail.com</email>; Tel.: +7-4956-237467; Fax: +7-4967-330553</corresp></author-notes><pub-date pub-type="epub"><day>01</day><month>1</month><year>2020</year></pub-date><pub-date pub-type="collection"><month>1</month><year>2020</year></pub-date><volume>10</volume><issue>1</issue><elocation-id>69</elocation-id><history><date date-type="received"><day>15</day><month>12</month><year>2019</year></date><date date-type="accepted"><day>30</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>© 2020 by the authors.</copyright-statement><copyright-year>2020</copyright-year><license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>We have shown that hydroxycobalamin (vitamin B<sub>12b</sub>) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + B<sub>12b</sub>. It was found that cell death induced by DDC + B<sub>12b</sub> differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B<sub>12b</sub> used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.</p></abstract><kwd-group><kwd>hydroxycobalamin</kwd><kwd>vitamin B<sub>12b</sub></kwd><kwd>diethyldithiocarbamate</kwd><kwd>synergism</kwd><kwd>cytotoxicity</kwd><kwd>endoplasmic reticulum</kwd><kwd>ER-stress</kwd><kwd>cytoplasm vacuolization</kwd><kwd>paraptosis-like cell death</kwd></kwd-group></article-meta></front><floats-group><fig id="biomolecules-10-00069-f001" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Vitamin B<sub>12b</sub> enhances the cytotoxic effect of DDC in subconfluent cultures of tumor cells. (<bold>a</bold>–<bold>d</bold>) Enhancement of the cytotoxic effect of DDC by 25 μM B<sub>12b</sub> toward HT1080, HT29, HEp-2, and A431 cells. (<bold>e</bold>) Dependence of the cytotoxic effect of the combination 1 mM DDC + 25 μM B<sub>12b</sub> against subconfluent cultures of HEp-2 cells on the exposure time. The components (B<sub>12b</sub> and DDC) were added simultaneously 24 h after cell seeding. The action of DDC + B<sub>12b</sub> was interrupted by replacing the culture medium with a fresh growth medium. The cytotoxicity was estimated 48 h after the addition of DDC and B<sub>12b</sub> (see Materials and methods). Incubation with 1 mM DDC (open circles) and with DDC + B<sub>12b</sub> (filled circles). The data are the means ± s.e.m. of five separate experiments.</p></caption><graphic xlink:href="biomolecules-10-00069-g001"></graphic></fig><fig id="biomolecules-10-00069-f002" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Vacuolization of HEp-2 cells at the stage of death initiation by the combination of 1 mM DDC + 25 μM B<sub>12b</sub>. (<bold>a</bold>) Control (<bold>b</bold>) after 5 h-incubation of cells with 1 mM DDC; (<bold>c</bold>) and (<bold>d</bold>) after 3- and 5-h incubations with DDC + B<sub>12b</sub>. The arrows point to vacuoles in cells treated with DDC + B<sub>12b</sub>. Phase contrast microscopy. (<bold>a’</bold>), (<bold>b’</bold>), (<bold>c’</bold>), and (<bold>d’</bold>):selected areas enlarged in inserts (<bold>a</bold>), (<bold>b</bold>), (<bold>c</bold>), and (<bold>d</bold>).</p></caption><graphic xlink:href="biomolecules-10-00069-g002"></graphic></fig><fig id="biomolecules-10-00069-f003" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Changes in the ultrastructure of HEp-2 cells caused by incubation for 4 h in the culture medium containing 1 mM DDC and 25 μM B<sub>12b</sub>. (<bold>a</bold>) Ultrastructure of a control cell. Selected areas enlarged in inserts a, b, and c. Scale bars: 3 μm in the overview, 1 μm in a and b, and 0.5 μm in c. (<bold>b</bold>) Ultrastructure of a cell with moderate changes after incubation. Scale bars: 2 μm in the overview and 1 μm in series. (<bold>c</bold>) Ultrastructure of a cell with severe changes after the incubation. For distinguishing the cell from the extracellular space, the plasma membrane of the cell is marked with a dashed line. Scale bars: 2 μm in the overview and 0.5 μm in the left insert and in series. N, nucleus; n, nucleolus; ER, endoplasmic reticulum; G, Golgi apparatus; m, mitochondria; g, glycogen granules; fl, secondary lysosomes; afl, autophagolysosomes; v, vacuole-like ER sacks.</p></caption><graphic xlink:href="biomolecules-10-00069-g003"></graphic></fig><fig id="biomolecules-10-00069-f004" orientation="portrait" position="float"><label>Figure 4</label><caption><p>The absence of signs of apoptosis during initiation of cell death by a combination of 1 mM DDC with 25 μM B<sub>12b</sub>. (<bold>a</bold>) Inhibition of the activity of caspase-3 in HEp-2 cells incubated with DDC + B<sub>12b</sub>. As a positive control, the recombinant protein izTRAIL significantly increased the activity of caspase 3. (<bold>b</bold>) The absence of internucleosomal DNA fragmentation in HEp-2 cells treated by the combination DDC + B<sub>12b</sub>. 1—untreated control cells; 2—cells after 24 h of incubation with a combination of 1 mM dithiothriethol + 25 μM B<sub>12b</sub>, which causes apoptosis [<xref rid="B18-biomolecules-10-00069" ref-type="bibr">18</xref>] (positive control); 3—cells after 24 h of incubation with DDC + B<sub>12b</sub>; 4—molecular weight markers. (<bold>c</bold>) The pan-caspase inhibitor zVAD.fmk did not protect cells from death induced by the combination DDC + B<sub>12b</sub>. zVAD.fmk (50 μM) was added to culture medium 1.5 h before the addition of DDC + B<sub>12b</sub> for 6 h, after which the medium was replaced with fresh one and then after 48-h cultivation the cytotoxicity was evaluated. The data are the means ± s.e.m. of three separate experiments.</p></caption><graphic xlink:href="biomolecules-10-00069-g004"></graphic></fig><fig id="biomolecules-10-00069-f005" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Confocal microscopy images of ER-mediated vacuolization of НЕр-2 (<bold>a</bold>–<bold>c</bold>) and A549 (<bold>d</bold>–<bold>f</bold>) cells after 6 h of incubation with 1 mM DDC + 25 µM B<sub>12b</sub>. (<bold>a</bold>), (<bold>d</bold>) control cells; (<bold>b</bold>), (<bold>e</bold>) cells incubated for 6 h with 1 mM DDC; (<bold>c</bold>), (<bold>f</bold>) cells incubated for 6 h with DDC + B<sub>12b</sub>. Staining with H342 (1 µg/mL), ER-Tracker Red (1 µM), and LysoTracker Green (0.2 µM).</p></caption><graphic xlink:href="biomolecules-10-00069-g005"></graphic></fig><fig id="biomolecules-10-00069-f006" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Absence of the signs of autophagy and the involvement of lysosomes in the induction of death of HEp-2 cells by the combination 1 mM DDC + 25 µM B<sub>12b</sub>. (<bold>a</bold>) Immunoblot analysis of autophagy markers in HEp-2 cells after the 1-h and 4-h incubation with DDC (1 mM) or DDC + B<sub>12b</sub>; (<bold>b</bold>) immunoblot analysis of autophagy markers in HEp-2 cells after the 4-h incubation in SGGP-deprived medium; (<bold>c</bold>) FL3 fluorescence of HEp-2 cells loaded with AO in untreated cultures, after 4-h starvation in SGGP-free medium, after 4-h incubation with DDC, B<sub>12b</sub>, and DDC + B<sub>12b</sub>; (<bold>d</bold>) The chelators of intracellular iron ions and autophagy inhibitors did not protect cells during the 6-h incubation with DDC + B<sub>12b</sub>. Cells were incubated in growth medium for 90 min with inhibitors or chelators, then DDC + B<sub>12b</sub> was added, and after 6-h incubation, medium was replaced by a fresh growth medium without additives. The cytotoxic effect was estimated 48 h after the treatment. *, differences between the samples and the control are significant, <italic>p</italic> < 0.05.</p></caption><graphic xlink:href="biomolecules-10-00069-g006"></graphic></fig><fig id="biomolecules-10-00069-f007" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Confocal microscopy images of HEp-2 cells stained with Mitotracker Green (0.2 µM), ER-tracker Red (1 µM), and the H342 (1 μg/mL). (<bold>a</bold>) Control; (<bold>b</bold>) cells after 5-h incubation with 1 mM DDC and (<bold>c</bold>) with 1mM DDC + 25 μM B<sub>12b</sub>. The membranes of vacuoles that appeared in cells were stained with red ER tracker but not with the green Mitotracker.</p></caption><graphic xlink:href="biomolecules-10-00069-g007"></graphic></fig><fig id="biomolecules-10-00069-f008" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Effect of 1 mM DDC and 25 µM B<sub>12b</sub> alone and of their combination on the indicators of mitochondrial activity: (<bold>a</bold>) capability of mitochondria to accumulate Ca<sup>2+</sup> in permeabilized cells; (<bold>b</bold>) the amount of intracellular ATP; (<bold>c</bold>) membrane mitochondrial potential evaluated by flow cytometry of cells loaded with 10 nM DiOC6(3); (<bold>d</bold>–<bold>f</bold>) confocal microscopy images of HEp-2 cells loaded with H342 (1 μg/mL) and 10 nM DiOC6(3); (<bold>d</bold>) control (untreated) cells; (<bold>e</bold>) cells after 4-h incubation with DDC; and (<bold>f</bold>) cells after 4-h incubation with DDC + B<sub>12b</sub>. The concentrations of myxothiazol (Myx) and oligomycin (Oligo) were 20 and 10 µM, respectively. *, differences between the sample and the control are significant, <italic>p</italic> < 0.05.</p></caption><graphic xlink:href="biomolecules-10-00069-g008"></graphic></fig><fig id="biomolecules-10-00069-f009" orientation="portrait" position="float"><label>Figure 9</label><caption><p>Effect of 1 mM DDC, 25 µM B<sub>12b</sub>, and of their combination on the cell redox balance. (<bold>a</bold>–<bold>c</bold>) Changes in the level of intracellular oxidized and reduced glutathione. At the indicated time, attached and detached HEp-2 cells were harvested and used for the determination of the content of GSH (<bold>a</bold>) and GSSG (<bold>b</bold>), and their ratio (<bold>c</bold>). Each measurement was repeated twice. (<bold>d</bold>) Antioxidants (catalase and thiols) partially inhibit the cytotoxic effect induced by 6-h incubation with DDC + B<sub>12b</sub>. * <italic>p</italic> < 0.05, compared to cells treated with DDC + B<sub>12b</sub>. (<bold>e</bold>), (<bold>f</bold>) Flow cytometry data on oxidative activity in cells loaded with 20 µM DCHFDA (<bold>e</bold>) and 1 µM MitoSOX (<bold>f</bold>). (<bold>g</bold>) and (<bold>h</bold>) Confocal microscopy images of HEp-2 cells stained with H342 and MitoSOX before incubation (<bold>g</bold>) and after 4-h incubation (<bold>h</bold>) with DDC+B<sub>12b</sub>. (<bold>i</bold>) Analysis of mBCl fluorescence in cells incubated with DDC and DDC + B<sub>12b</sub>. After incubation, cells were washed twice and stained with 60 μM mBCl. Cells incubated with 5 mM ClDNB (10 min) served as a negative control. *, differences between samples and the control are significant, <italic>p</italic> < 0.05; #, differences between samples are significant, <italic>p</italic> < 0.05.</p></caption><graphic xlink:href="biomolecules-10-00069-g009"></graphic></fig><fig id="biomolecules-10-00069-f010" orientation="portrait" position="float"><label>Figure 10</label><caption><p>ER stress induced by the action of DDC + B<sub>12b</sub> causes the disturbance of the synthesis and folding of the protein in HEp-2 cells. Immunoblot analysis of ER-stress markers in HEp-2 cells after 1-h and 4-h incubations with DDC (1 mM) or DDC (1 mM) + B<sub>12b</sub> (25 µM) (<bold>a</bold>). *, differences are significant compared with control cells; #, significant differences between the groups, <italic>p</italic> < 0.05. The effect of protein synthesis inhibitors on the cytotoxicity of DDC + B<sub>12b</sub> (<bold>b</bold>) and vacuolization (<bold>c</bold>). Cells were incubated in growth medium for 90 min with 4-PBA or CHX, then DDC + B<sub>12b</sub> was added, and after 5-h incubation, medium was replaced by a fresh growth medium without additives. The vacuolization was estimated immediately (bright field), and the cytotoxic effect was estimated 48 h after the treatment. *, differences are significant compared to cells incubated without the inhibitors, <italic>p</italic> < 0.05. The arrows point to vacuoles in cells treated with DDC + B<sub>12b</sub>.</p></caption><graphic xlink:href="biomolecules-10-00069-g010"></graphic></fig><fig id="biomolecules-10-00069-f011" orientation="portrait" position="float"><label>Figure 11</label><caption><p>Increase in the concentration of intracellular Ca<sup>2+</sup> during the initiation of cell death by the combination of 1 mM DDC + 25 μM B<sub>12b</sub> and the effect of the inhibitor of IP3 receptor and chelator of extracellular calcium on the cytotoxicity of the combination. (<bold>a</bold>) and (<bold>b</bold>) FL1 fluorescence of cells stained with 5 μM Fluo-4AM after incubation with DDC and B<sub>12b</sub>. (<bold>c</bold>) Effect of the IP3 receptor inhibitor and EGTA on the initiation of cell death by the combination DDC+B<sub>12b</sub> and cell vacuolization (<bold>d</bold>–<bold>i</bold>). Cells were incubated in growth medium for 90 min with 20 μM 2-APB or with 2.5 mM EGTA, then DDC + B<sub>12b</sub> was added, and after 6-h incubation, medium was replaced by a fresh growth medium without additives. Cytotoxicity was estimated 48 h after the treatment as compared with the control. *, <italic>p</italic> < 0.05, compared to cells treated with DDC + B<sub>12b</sub>. The vacuolization of HEp-2 cells was estimated 6 h after the addition of DDC+B<sub>12b</sub> (bright field). Control (<bold>d</bold>), 2.5 mM EGTA (<bold>e</bold>), 20 μM 2-APB (<bold>f</bold>); DDC + B<sub>12b</sub> (<bold>g</bold>), EGTA + DDC + B<sub>12b</sub> (<bold>h</bold>), and 2-APB + DDC + B<sub>12b</sub> (<bold>i</bold>). The arrows indicate vacuoles.</p></caption><graphic xlink:href="biomolecules-10-00069-g011"></graphic></fig><fig id="biomolecules-10-00069-f012" orientation="portrait" position="float"><label>Figure 12</label><caption><p>A hypothetic scheme of intracellular events during the initiation of paraptosis-like tumor cell death by the combination DDC + B<sub>12b</sub> (for details, see the text).</p></caption><graphic xlink:href="biomolecules-10-00069-g012"></graphic></fig><table-wrap id="biomolecules-10-00069-t001" orientation="portrait" position="float"><object-id pub-id-type="pii">biomolecules-10-00069-t001_Table 1</object-id><label>Table 1</label><caption><p>IC<sub>50</sub> values for DDC used alone and in combination with 25 μM B<sub>12b</sub>, and CI for different cell lines</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Cell Line</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">IC<sub>50</sub> for DDC Alone, mM</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">IC<sub>50</sub> for DDC Combined with B<sub>12b</sub>, mM</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Chou-Talalay Combination Index</th></tr></thead><tbody><tr><td align="center" valign="middle" rowspan="1" colspan="1">A549</td><td align="center" valign="middle" rowspan="1" colspan="1">2.47 ± 0.64 *</td><td align="center" valign="middle" rowspan="1" colspan="1">0.4 ± 0.05 *</td><td align="center" valign="middle" rowspan="1" colspan="1">0.17</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1">A431</td><td align="center" valign="middle" rowspan="1" colspan="1">1.86 ± 0.21 *</td><td align="center" valign="middle" rowspan="1" colspan="1">0.43 ± 0.04 *</td><td align="center" valign="middle" rowspan="1" colspan="1">0.24</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1">HEp-2</td><td align="center" valign="middle" rowspan="1" colspan="1">2.22 ± 0.24 *</td><td align="center" valign="middle" rowspan="1" colspan="1">0.46 ± 0.09 *</td><td align="center" valign="middle" rowspan="1" colspan="1">0.22</td></tr><tr><td align="center" valign="middle" rowspan="1" colspan="1">HT1080</td><td align="center" valign="middle" rowspan="1" colspan="1">4.79 ± 0.94</td><td align="center" valign="middle" rowspan="1" colspan="1">0.5 ± 0.07</td><td align="center" valign="middle" rowspan="1" colspan="1">0.11</td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">HT29</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">2.33 ± 0.42</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">0.53 ± 0.06</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">0.24</td></tr></tbody></table><table-wrap-foot><fn><p>* The results have been reported earlier [<xref rid="B38-biomolecules-10-00069" ref-type="bibr">38</xref>].</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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