Triggering ROS-production is one of the major tumoricidal mechanisms of gemcitabine as pancreatic cancer cells with lower basal ROS-levels are more resistant to gemcitabine (Dalla Poza et al. 2012). Gemcitabine-activation of acid sphingomyelinase also triggers insertion of proapoptotic bax protein into the mitochondrial membrane and strongly induces death of glioma cells (Dumitru et al. 2009).

All these data are compatible with our findings showing damage and reduction of mitochondria with gemcitabine treatment in C6 cells.   Gemcitabine Induction of Gutta Adipis / Lipid Droplets. Likely Result of Reduced Phospholipids and Impaired Mitochondrial Activity   In our study, we observed formation of lipid droplets / gutta adipis with gemcitabine, which was enhanced with COX-inhibitors. Lipid droplets are not stagnant depot sites, rather they are dynamic metabolic organelles, with their abundance coupled to the cellular metabolic state (Kory et al. 2016).

The neutral lipids (triglycerides and sterol esters) stored in their cores provide a storage for membrane lipids and protect against energy fluctuations. Their monolayer surface has a lipid composition similar to the ER phospholipids. Their number and size are determined by the amount and type of phospholipids available to cover their surface. When sufficient phospholipids are not available to cover their surface, surface tension increases leading their fusion. Lipid droplet-coalescence increases in cells with defects in phospholipid synthesis (Kory et al. 2016). Within the skeletal myocytes, lipid droplets act as fuel depots for mitochondrial fat oxidation and usually juxtapose mitochondria to allow fast transport (Stephens et al. 2011).

With ageing, the numbers of lipid droplets increase yet their association with mitochondria are disrupted (Stephens et al. 2011). Gamma-linolenic acid (GLA) inhibits Walker 256 tumor growth in vivo and doubles the triacylglycerol and lipid droplet-content within the tumor cells (Colquhoun et al. 2002).

Simultaneously, the surface density of mitochondrial cristae reduces, along with reductions in contact sites and matrix granules. Enhancement of lipid droplets were also defined in cancer cells exposed to biological antitumor agents or antitumor microRNA. In renal carcinoma cells, miR-494 expression markedly increased multilamellar bodies and lipid droplets and reduced cell viability accompanied by increased cleaved PARP and autophagic LC3B protein (Dutta et al. 2016).

Besides autophagy, LC3B involves in the formation of lipid droplets; and its conjugated form LC3-II associates with the phosphatidylethanolamine of the lipid droplet-membrane (Dutta et al. 2016). Formation of lipid droplets accompanies mitochondrial changes including their shortening, fragmentation and becoming more electron-dense (Dutta et al. 2016), which are exactly parallel to our findings in gemcitabine-treated C6 cells. In cells exerting simultaneous enhancement of autophagy and lipid droplets, a recycling process may occur which involves concurrent lipid accumulation and utilization, as lipid droplet digestion occurs via autophagy (Zhao et al. 2014). Gemcitabine enhancement of lipid droplets may be due to: 1- Impairment of mitochondrial metabolism, 2- Shortage of phospholipids, 3- A lipid-recycling attempt of cancer cells to direct lipids to cellular regions, where lipid structures are damaged following chemotherapy.   Gemcitabine-Induced Enlargement of ER Cisternae and Formation of ER Whorls.

Collapsed ER Whorls With Gemcitabine+DMSO Treatment   In our study, ER whorls surrounded pyknotic nuclei in gemcitabine treated C6 glioma cells. In gemcitabine+DMSO co-treated groups, cisternal enlargement of ER membranes and formations of stacks were seen; while in some cells, ER membranes formed whorls which collapsed within themselves. ER is the major site of protein synthesis, folding and modification. Noxious events including oxidative stress and glucose deprivation can damage the ER which causes accumulation of disrupted and misfolded proteins in the ER lumen, which leads to ER stress (Cheng et al. 2015). Two protective strategies exist to alleviate the ER stress, the heat shock response (HSR) and the unfolded protein response (UPR), which provide quality of protein folding and inhibits protein synthesis.

HSR induces molecular chaperones to reduce the misfolded proteins and UPR is induced by three ER receptors: activating transcription factor-6 (ATF6), inositol-requiring enzyme-1 (IRE1) and PKR-like ER kinase (PERK) (Cheng et al. 2015). PERK has the main role to cope with mild ER stress and triggers the UPR: decreased protein synthesis pathway (Cheng et al. 2015). Very noteworthy, IRE1 directly involves in proliferation and gemcitabine-resistance of pancreatic cancer cells (Chien et al. 2014). Studies in 14 pancreatic cancer cells revealed that various IRE1-inhibitors induced apoptosis and sensitized to gemcitabine (Chien et al. 2014).

Gemcitabine induced ER-cisternal enlargement may be a protective factor against chemotherapy stress and to shunt aminoacids to be utilized in energetic pathways. ER whorls which collapse within themselves develop in several conditions: 1- Nutritional starvation, hormonal starvation and blockage of anabolic metabolism (Price et al. 1977; Taira et al. 1981; Walsh et al. 1982; Kubasik-Juraniec et al. 1999), cellular stress (Kou et al. 1974; Dabholkar and Carmichael 1987; Garthweie et al.

1992), 2- Inhibition of protein (Kou et al. 1974) or cholesterol synthesis (Lum and Wright 1995), 3- Disruption of ER transport and Golgi Systems causing accumulation of large lipid droplets (Sanguinetti et al. 1995; Zhou et al. 2011), 4- Carcinogenesis (de Nicola et al.

1978; Wessely et al. 1982, Muakkassah-Kelly et al. 1988; Moroni and Barbatelli 2001); 5- Germ cell development (Gremigni and Nigro 1983; Kallenbach 1984; Werner and Moutairou 2000).

ER whorls occur in pancreatic cells during fasting and demonstrated as inactive RER lamellae (Taira et al. 1981). Fasting induces ER-whorls in Mosqiuto intestinal epithelia whereas feeding with blood and subsequent aminoacid supply causes robutst unwinding of ER (Zhou et al. 2011). Loss of ER protein Yip1A, providing proper ER/golgi membrane networks, or decrease of alpha-COPI coatomer protein induced collapse of ER structures into whorls (Zhou et al. 2011).

Cancer cells may not prefer to synthesize new proteins under stress conditions; instead they shunt aminoacids into pathways to provide energetic fuel. Indeed, polyribosomal profiling in pancreatic cancer cells showed that gemcitabine induced pan- suppression of translation (Palam et al. 2015). Prominent induction of ER-whorls in C6 cells with gemcitabine and gemcitabine+DMSO treatments strongly suggest that these agents induced shortage of essential cellular molecules.   Conclusions   Comprehensive ultrastructural analysis of cancer cell responses against antitumor treatments may provide a deeper view on the action mechanisms of antineoplastic drugs. A best approach would be combining old and new methods, in which gene expression data are interpreted in the context of electron microscopical ultrastructural findings. In our future studies, we will repeat these studies in human glioma cells and interpret these findings with simultaneously applied microarray data. Gemcitabine and vinorelbine induced both overlapping and distinct fine structural actions within C6 glioblastoma tumor cells and understanding these mechanisms may help to develop better combinatorial strategies not just for glioma treatment but also for other types of malignancies, where their clinical efficacies are proven.


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