Bis (3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine photodynamic therapy induces in vivo tumor ablation by triggering cytochrome c dependent mitochondria mediated apoptosis
Authors: Annie Abraham
D Gayathri Devi
T R Cibin
Journal: Photodiagnosis and Photodynamic Therapy (2013)
Discipline: CLINICAL MEDICINE
Keywords: 7,12-Dimethyl benzanthracene; Apoptosis; Photodynamic therapy; Skin tumor; Squaraine dyes.
Affiliations: Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695581, Kerala, India.

Abstract:  Background: Despite findings that photodynamic treatment with bis (3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine initiated tumor regression in mice skin, queries regarding its mode of action - answers to which will be functional to design clinical trials on squaraine based photodynamic therapy - remain unanswered. Our investigation reveals the in vivo mechanism of action of the photosensitizer. Methods: Skin tumor was induced in Swiss albino mice using 7,12-dimethyl benzanthacene. After the intraperitoneal administration of the dye in tumor induced mice, its concentration in subcellular fractions of the tumor tissue was determined fluorimetrically. Cytochrome c release from the mitochondrial membrane after the photodynamic treatment was analyzed. The observations stemming from this part lead to histopathological examination of tumor tissues. Apoptotic markers like caspase-3, Bcl-2 and Bax were also studied. Results: Major portion of the dye accumulated in the mitochondria. Cytochrome c leakage from mitochondria after squaraine PDT suggests loss of mitochondrial membrane integrity, which was further confirmed by the results of histopathological analysis. The activity of caspase-3 was elevated, expression of Bcl-2 diminished and that of Bax increased - all these results show enhancement of apoptosis in the tumor region after the treatment. Conclusions: The results lead to the elucidation of mechanism of tumor destruction which proves to be mitochondria mediated apoptotic damage of tumor tissue. The study assumes significance since it defines the in vivo mode of action of a photosensitizer. Also, the query of how a squaraine based photosensitizer evokes tumor response is being dealt with here, for the first time. 

Retracted: No

PubPeer comments:
 Images in Figure 4 appear to include some repeated elements. ***  , 
 In Figure 2, the tissue samples for Group II and Group IV appear unexpectedly similar, as if they represented serial slices of the same animal. ***  , 
 Response for comment 1 The marked regions in pink could be background which are not part of the bands. It may have occurred during developing of the nitrocellulose membrane. The marks could also be from the membrane forceps, as the same forceps were used for all the western blots. The regions marked in green and blue are not identical. The background, length, width and inclination of the bands are entirely different. The differences can be easily identified on first observation itself. Also, the bands in question are of the loading control (uf062-actin), which could of the same intensity if equal amount of proteins are loaded. Response for comment 2 The histological examination was not done in our laboratory since our Department Institute did not have enough facilities for that. The sectioning, staining and imaging was done by an expert in a diagnostic laboratory. We have represented the results as obtained from the diagnostic laboratory. Tissue sections were prepared separately for different groups. Even though the alleged region has some similarities, it may be due to similar distribution of cellular components, lesions and ultra-structure of the tumor region. Several differences can be noted, like the visible keratinisation in the tissue of group II animals, which is not seen in Group IV. , 
 The marked regions in pink could be background which are not part of the bands. It may have occurred during the developing of the nitrocellulose membrane. The marks could also be from the membrane forceps, asthe same forceps were used for all the western blots.The regions marked in green and blue are not identical.The background, length,width and inclination of the bands are entirely different. The differences can be easily identified on first observation itself.Also, the bands in question are of the loading control (beta-actin), which could of the same intensity if equal amount of proteins are loaded. , 
 #2 The histological examination was not done in our laboratory since our Department Institute did not have enough facilities for that. The sectioning, staining and imaging was done by an expert in a diagnostic laboratory. We have represented the results as obtained from the diagnostic laboratory. Tissue sections were prepared separately for different groups. Even though the alleged region has some similarities, it may be due to similar distribution of cellular components, lesions and ultra-structure of the tumor region. Several differences can be noted, like the visible keratinisation in the tissue of group II animals, which is not seen in Group IV. , 
 Thank you for your replies. Regarding the concerns in Figure 4, your hypothesis for why the images might appear as they do is certainly possible, if not convincing. A more simple explanation would account for the background similarities (also evident in Figure 4); I can elaborate if necessary. However, if the authors can provide the original uncropped scans, that will put this issue to rest. If those are unavailable, I can demonstrate my concerns more definitively. Regarding the concerns in Figure 2, again your conjecture for why the images might appear as they do is certainly possible. As the experiment was not performed by your lab, it seems as if you cannot directly address my concern that these are more likely to be serial samples from the same animal. To help reassure readers, could you provide the contact information for the lab, the original data they supplied, and perhaps consider adding this disclosure as a corrigendum to inform readers (apologies if this is described in the methods, I did not see it). , 
 I agree with Actinopolyspora s concerns. Figure 2 panels from this paper also share a remarkable similarity to panels in Figure 11 of another paper by this group, published in 2010, i.e. T.R. Cibin et al., Phytother. Res. 24 (2010), DOI: 10.1002 ptr.2950 - https: pubpeer.com publications 0C34E2DA9E62D02112C19AA9DF2D6E There they represent the controls of a different experiment. Could the authors please clarify the similarities, which I have marked with blue and green boxes?  ,